Clathrin coats are involved in a variety of transport processes,

Save this PDF as:
 WORD  PNG  TXT  JPG

Size: px
Start display at page:

Download "Clathrin coats are involved in a variety of transport processes,"

Transcription

1 Clathrin-coated pits with long, dynamin-wrapped necks upon expression of a clathrin antisense RNA T.-G. Iversen*, G. Skretting*, B. van Deurs, and K. Sandvig* *Institute for Cancer Research at the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway; and Structural Cell Biology Unit, Department of Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark Edited by Pietro V. De Camilli, Yale University School of Medicine, New Haven, CT, and approved January 14, 2003 (received for review July 15, 2002) To investigate the role of clathrin in coated vesicle formation, a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA was produced. After 18 h of CHC antisense RNA expression, the internalization of transferrin was inhibited by 90%. Although the amount of CHC was reduced by only 10%, the frequency of clathrin-coated pits at the cell surface increased by a factor of 3 5, and clathrin-coated structures also accumulated on a pleiomorphic, multivesicular, endosomal compartment. Remarkably, the coated pits were connected to the cell surface by long, tubular necks wrapped by dynamin rings, and the level of dynamin in the CHC antisense RNA-expressing cells was up-regulated 10- fold. In contrast, the amount of several other proteins associated with clathrin coat formation was unaffected. Thus, this study demonstrates that CHC antisense RNA causes accumulation of clathrin-coated pits with dynamin rings around the neck in intact cells not transfected with dynamin mutants, suggesting the existence of a previously uncharacterized functional interplay between clathrin and dynamin. Clathrin coats are involved in a variety of transport processes, and different ways to interfere with clathrin-dependent steps are of interest in elucidating mechanisms of coat and vesicle formation and in clarifying functions of clathrin coats on various organelles. At the plasma membrane, clathrin-coated pits are involved in uptake of receptors and ligands. Clathrin also is present in the trans-golgi network and on lysosomes and endosomes (1, 2). In the perinuclear recycling compartment, clathrin may be involved in both transport to the Golgi apparatus (3) and transferrin receptor recycling (4). Flat clathrin-coated microdomains could function to sort ubiquitinated membrane proteins destined for degradation (5, 6). Furthermore, clathrin may be important for early endosome distribution (7). The exact mechanism(s) involved in formation of clathrincoated vesicles is not known, but molecules such as dynamin, endophilin, and epsin (8) all might be involved. Several approaches have been used to inhibit vesicle formation from clathrin-coated structures (9). Recently, overexpression of mutant proteins or protein domains has been used to block clathrindependent endocytosis, but this method is likely to affect more than one function. For instance, overexpression of mutant dynamin (K44A) will inhibit not only uptake from clathrincoated pits but also other types of endocytosis (10). Futhermore, overexpression of protein domains might lead to low-affinity interactions that normally do not occur, thereby depleting the cytosol of interacting proteins required for other functions. It is therefore useful to apply methods based on different principles. To investigate the importance of clathrin, we have established a cell line with inducible expression of clathrin heavy chain (CHC) antisense RNA. In the present article we demonstrate some unexpected results concerning regulation of dynamin levels and accumulation of clathrin-coated structures. Materials and Methods Materials. Geneticin (G418) was obtained from Serva. Tetracycline (tet), puromycin, pronase, aprotinin, transferrin, Hepes, Na Mes, BSA, and lactose were obtained from Sigma. Na 125 I and Expre 35 S 35 S Protein Labeling Mix were obtained from Perkin Elmer. Protein A Sepharose, protein G agarose, and [ 32 P]dCTP were obtained from Amersham Biosciences. Transferrin was labeled by the iodogen method (11) to a specific activity of 20,000 30,000 cpm ng. ImmunoPure NHS-SS-Biotin was obtained from Pierce, and Dynabead M-280 Streptavidin was obtained from IGEN International (Gaithersburg, MD). The following antibodies were used: goat anti-chc (Santa Cruz Biotechnology), mouse anti-chc (Research Diagnostics, Flanders, NJ), mouse anti- -adaptin (AP2; Affinity BioReagents, Golden, CO), mouse anti-adaptin (AP1) and anti-tubulin (Sigma), anti-cathepsin D (Zymed), anti-dynamin (Hudy 1; Upstate Biotechnology, Lake Placid, NY), anti-amphiphysin I (PharMingen), and anti-epidermal growth factor receptor pathway substrate clone (EPS)-15 and anti-clathrin light chain (Covance Research Products). Anti-epsin and anti-endophilin were a kind gift from P. De Camilli (Yale University, New Haven, CT). The cdna for CHC was generously provided by T. Kirchhausen (Harvard University, Boston). The plasmid containing hemagglutinin-tagged dynamin 2 wild type was kindly provided by S. L. Schmid (The Scripps Research Institute, La Jolla, CA). Cells. The BHK21-tTA cell line, kindly provided by A. Wandinger-Ness (University of New Mexico School of Medicine, Albuquerque) (12), was grown in complete DMEM (Flow Laboratories) supplemented with 7.5% FCS, 2 mm L-glutamine, 100 units ml penicillin, 100 g ml streptomycin, and 200 g ml geneticin. Stable BHK21-tTA anti-chc cells were maintained in select DMEM (complete DMEM containing 200 ng ml puromycin and 2 g ml tet). For induction of CHC antisense RNA expression, tet was removed from the medium. Plasmid Constructs. The entire coding region of rat CHC cdna (13) was cloned into plitmus (New England Biolabs) and, further, into the expression plasmid ptet-splice (GIBCO BRL) in the antisense orientation, giving the construct pachc. Generation of a Stable Cell Line. The BHK21-tTA anti-chc cell line was generated by transfection of subconfluent BHK21-tTA cells (12) with 10 g of pachc and 1 g of the plasmid pbspac, which contains the puromycin resistant gene (14), by using the transfection reagent DOTAP (Roche Molecular Biochemicals) according to the manufacturer s instructions. After transfection, the cells were allowed to recover in complete DMEM for 24 h before passage and transfer to complete DMEM containing 1 g ml puromycin. Individual BHK21-tTA anti-chc clones were isolated, and transferrin endocytosis was measured. A This paper was submitted directly (Track II) to the PNAS office. Abbreviations: CHC, clathrin heavy chain; tet, tetracycline. See commentary on page T.-G.I. and G.S. contributed equally to this work. Present address: Norwegian School of Veterinary Science, N-0033 Oslo, Norway. To whom correspondence should be addressed. CELL BIOLOGY SEE COMMENTARY cgi doi pnas PNAS April 29, 2003 vol. 100 no

2 selected clone of BHK21-tTA anti-chc was subcloned by limited dilution. Western Blot Analysis. For analysis of clathrin and tubulin (as internal standard), cells were scraped off the plastic and lysed in Mes buffer (0.1 M Mes, ph mm MgCl 2 1 mm EGTA 20 M leupeptin 1 mm PMSF 0.2% Triton X-100). Samples were resolved on a 7.5% SDS PAGE gel under reducing conditions. The proteins were transferred to nitrocellulose (Schleicher & Schuell), and the membrane was blocked with 5% nonfat dry milk in PBS containing 0.05% Tween 20. For analysis of the different accessory proteins playing a role in clathrin-coated pit formation, cells were solubilized in RIPA buffer (50 mm Tris HCl, ph mm NaCl 1% deoxycholate 0.1% SDS 1.5% Triton X-100) supplemented with the Complete protease inhibitor mixture (Roche Diagnostics). Samples were resolved on % SDS PAGE gels, and the proteins were transferred to poly(vinylidene difluoride) membranes (Millipore). The filters were incubated with primary antibodies in blocking solution for 1hatroom temperature, washed three times for 10 min with 0.05% Tween 20, and finally incubated with secondary antibodies conjugated to horseradish peroxidase for 45 min at room temperature. The results were developed with a chemiluminescent detection kit (Pierce). Metabolic Labeling of Cells and Immunoprecipitation. The cells were starved for 30 min in DMEM lacking Met Cys. [ 35 S]Met [ 35 S]Cys (Expre 35 S 35 S label) was added to the medium (75 Ci ml; 1 Ci 37 GBq), and the cells were incubated for another 30 min. The cells were then scraped off and lysed in a Mes buffer containing 0.2% Triton X-100 (see Western Blot Analysis). The cell lysate was aspirated six times through a 21-gauge needle, incubated on ice for 20 min, and centrifuged at 6,800 g for 10 min at 4 C. The protein concentration in the supernatant was determined by using the Protein Assay kit (Bio-Rad). One hundred micrograms of total protein was immunoprecipitated with goat anti-chc (30 l) bound to protein G agarose (25 l) for 24 h at 4 C on a rotating platform. Immunocomplexes were washed four times with 500 l oftbs buffer (50 mm Tris, ph M NaCl), resuspended in sample buffer, and separated on a 7.5% SDS PAGE gel under reducing conditions. For degradation experiments, cells were plated and incubated overnight before they were transferred to Met- and Cys-free medium. A mixture of [ 35 S]Met [ 35 S]Cys (10 Ci ml) was added, and the cells were incubated for another 24 h in the presence of tet. The cells then were incubated in normal medium without tet, in the presence or absence of lactacystin (10 M) for 24 h. Cell lysates were prepared, and CHC was immunoprecipitated, as described above. Radioactive bands were visualized by fluorography, and quantification was performed by using a scanning densitometer. The rate of dynamin synthesis was measured by a metabolic pulse labeling experiment. Cells grown for 2 days with or without tet were Met Cys-starved for 30 min. Then they were pulsed with [ 35 S]Met [ 35 S]Cys (75 Ci ml) for 30 min and chased in complete DMEM 10% FCS for 10 min. The cells were lysed in RIPA buffer and centrifuged at 3,800 g for 10 min. Cell lysates were incubated with 20 l of protein A Sepharose prebound to anti-dynamin (5 l of Hudy 1). Immunocomplexes were washed with RIPA buffer and resolved by using a 10% SDS/PAGE gel. The gel was exposed to PhosphorImager screens and analyzed by using PHOSPHORIMAGER and IMAGEQUANT software (Molecular Dynamics). Measurements of Endocytosis and Recycling. Endocytosis of transferrin was measured after a 5-min incubation at 37 C with 125 I-transferrin ( 100 ng ml) in Hepes medium. The cells were washed with ice-cold Hepes and then treated with pronase (2 mg ml) in Hepes medium for 1 h on ice. The cells were removed from the incubation medium by centrifugation, and the radioactivity in the cell pellet (endocytosed transferrin) and in the supernatant (surface-bound transferrin) was measured. Recycling of transferrin was measured by using TAG- and biotinlabeled transferrin as described (15). Sorting and Processing of Cathepsin D. The assay was performed as described (25): The cells were starved for Met Cys for 30 min, pulsed with [ 35 S]Met [ 35 S]Cys for 30 min, and chased for various time periods in the presence of mannose-6-phosphate (5 mm) to inhibit endocytosis of released enzyme. Metabolically labeled cathepsin D was immunoprecipitated from cell lysates and media. The relative amounts of procathepsin D and mature forms of cathepsin D were determined by SDS PAGE, autoradiography, and densitometry. Electron Microscopy. The cells were washed with PBS and fixed with 0.1% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, ph 7.2, followed by dehydration and embedding in Epon. Alternatively, the fixed cells were processed for ultracryosections and subsequent ImmunoGold labeling to localize dynamin (16). In some experiments, cells were fixed in the presence of ruthenium red (0.5 mg ml) before they were washed and embedded in Epon (16). Finally, some cells were incubated for 1 h at 37 C with horseradish peroxidase (5 mg ml; Sigma type II) before fixation as described (16). Sections were analyzed with a Philips 100 CM electron microscope (Eindhoven, The Netherlands). RNA Preparation and Northern Blot Analysis. Total RNA was isolated by using the RNeasy mini kit (Qiagen), was run on a 1% agarose gel with formaldehyde, and finally was blotted onto a Hybond-N membrane (Amersham Biosciences). The probe for dynamin 2 detection was generated by labeling the entire cdna of dynamin 2 with Redivue [ - 32 P]dCTP by using the Random Primers DNA labeling system (Invitrogen). Hybridization was done overnight at 60 C. Results and Discussion Synthesis of CHC Is Reduced in Cells Expressing CHC Antisense RNA. Here we have used a tet-controlled expression system that is tightly regulated (17) to produce BHK cells with inducible expression of CHC antisense RNA. The maximum level of expression of the inserted gene in this system is 48 h (12, 18). The synthesis of CHC in different experiments was reduced by 25 40% after 48 h of CHC antisense RNA expression (Fig. 1A). As shown by Western blot analyses, the protein level was reduced by 50% after 2 days of induction, and after 4 days of induction the level was reduced to 28% (Fig. 1B). In this experiment we used an anti-chc that recognized an epitope close to the C-terminal end of the protein. The reduction of CHC was similar when an anti-chc against the N-terminal part of the protein was used (data not shown). After 24 h of induction, a reduction in the CHC level was barely detectable, a finding that agrees with earlier results demonstrating that the half-life of CHC is rather long (19). Furthermore, total RNA was extracted from cells grown in the absence or presence of tet for 2 and 4 days. After 2 days, the reduction in the endogenous CHC mrna level was 50% of control cells. After 4 days there was no detectable CHC mrna (data not shown). Transferrin Trafficking in CHC Antisense RNA-Producing Cells. Transferrin endocytosis was inhibited significantly after 16 h of CHC antisense RNA expression, and after 48 h the transferrin endocytosis was reduced by 90 95% (Fig. 1 C and D). This level remained constant when the cells were incubated for up to 6 days cgi doi pnas Iversen et al.

3 Fig. 1. Effect of CHC antisense RNA on clathrin and clathrin-dependent trafficking in the BHK21-tTA anti-chc cells. (A) Cells grown with or without tet for 2 days were pulse-labeled with [ 35 S]Met [ 35 S]Cys for 30 min and solubilized. The lysates were immunoprecipitated with goat anti-chc, and the precipitates were analyzed by SDS PAGE (7.5% gel) and autoradiography. (B) Cells were grown in the absence of tet for indicated time points and lysed, and equal volumes of the lysates were separated on a 7.5% SDS PAGE gel. The gel was subjected to Western blot analysis probing with goat anti-chc antibody and with anti-tubulin antibody (as internal standard). The film was scanned by densitometry and normalized against tubulin. (C) Cells were grown in the absence of tet for indicated time points and transferred to Hepes medium. 125 I-transferrin was added to the cells and endocytosed for 5 min. (D) Cells were grown with or without tet for 2 days. Then the endocytosis of 125 I-transferrin for the indicated times was measured. (E) The cells were [ 35 S]Cys [ 35 S]Met-labeled for 24 h in the presence of tet and further incubated in medium without tet and lactacystin for 24 h. CHC was immunoprecipitated with anti-chc, and the precipitates were analyzed on a 7.5% SDS PAGE gel. (F) Cells were induced to express CHC antisense RNA for 24 h lactacystin, and 125 I-transferrin endocytosis during a 5-min incubation at 37 C was measured. (G) Cells were grown with or without tet for 2 days and metabolically labeled. At the indicated chasing times the cells were lysed, and cathepsin D was immunoprecipitated from the lysate and the medium. The precipitates were analyzed on a 12.5% SDS PAGE gel. The gel then was subjected to autoradiography and densitometric quantification. CELL BIOLOGY SEE COMMENTARY (data not shown). The inhibition of transferrin endocytosis was accompanied by an increased binding of transferrin (2- to 3-fold) at the plasma membrane (data not shown), suggesting that recycling of the internalized transferrin receptor continued although the uptake became blocked. The rapid inhibition of transferrin uptake could be due to partial cleavage of clathrin molecules that might interfere (possibly in a dominant negative manner) with the formation of clathrin-coated vesicles. We therefore investigated whether lactacystin, an inhibitor of proteolysis, could counteract degradation of clathrin and the inhibition of transferrin endocytosis, and indeed this was the case. A pulse chase experiment showed that incubation with lactacystin decreased the degradation of clathrin (Fig. 1E). Western blot analysis revealed an increased clathrin level when cells were grown without tet and in the presence of lactacystin (data not shown). Furthermore, the reduction in transferrin endocytosis was partially abrogated by lactacystin (Fig. 1F). Lactacystin is an inhibitor of proteasomes (20) and of a newly characterized giant protease, tripeptidyl peptidase II (TPPII), possessing both the ability to remove tripeptides from the N terminus and an endoprotease activity (21). Although the effect of lactacystin might be indirect, the data are compatible with the idea that proteolytic processing of preexisting clathrin molecules might be involved in inhibition of clathrin-dependent endocytosis. Effect of CHC Antisense RNA Expression on Transport and Maturation of Cathepsin D. The lysosomal hydrolase cathepsin D is normally transported by a clathrin-dependent pathway from the Golgi apparatus to endosomes, where it is processed to a mature form. To test whether this pathway was affected by expression of CHC antisense RNA, we studied the kinetics of procathepsin D maturation. Induced expression of CHC antisense RNA inhibited the formation of mature cathepsin D by 50% after a 4-h chase (Fig. 1G), and there was a 5-fold increase in extracellular secretion of procathepsin D (Fig. 1G). Furthermore, the rate of procathepsin D synthesis was increased 2-fold. Lysosomes seemed normal by electron microscopy after 2 days of CHC antisense RNA induction, probably because of the high stability of lysosomal enzymes. However, 4 days of induction led to formation of very large lysosomes containing a lot of internal membrane (data not shown). Their formation is most likely a consequence of impaired transport maturation of lysosomal enzymes. Iversen et al. PNAS April 29, 2003 vol. 100 no

4 Fig. 2. Representative examples of clathrin-coated vesicular structures at the cell surface of BHK21-tTA anti-chc cells expressing CHC antisense RNA for 2 4 days. The clathrin-coated structures often form clusters. Moreover, they are frequently connected to the cell surface by long tubules. These tubules show a cross-striation (indicated in G and H). Arrowheads in A and B show the characteristic honeycomb-like pattern of tangentially sectioned clathrin-coated pits vesicles. (Bar 200 nm.) CHC Antisense RNA Leads to Accumulation of Clathrin-Coated Pits at the Cell Surface and on Endosomes. The effect of CHC antisense RNA expression on clathrin-associated structures at the plasma membrane and on endosomes was examined at the ultrastructural level. Numerous clathrin-coated pits and vesicular profiles were noticed at the cell surface (Fig. 2). The clathrin coats appeared structurally normal, even when cut tangentially, where the characteristic honeycomb-like pattern was evident (Fig. 2 A and B). Their diameter varied from 80 to 150 nm. They often formed aggregates (Fig. 2 C E) and appeared to be connected to the cell surface by long tubules (necks) with a diameter of nm. These tubules often showed a cross-striation with a periodicity of 20 nm (Fig. 2). It turned out that virtually all clathrin-coated profiles at the cell surface were indeed connected to the cell surface, because they were stained by ruthenium red added during fixation (Fig. 3 A and B). Small vesicular profiles with a diameter of 30 nm were often also stained by ruthenium red. These may represent cross-sectioned tubules. There appeared to be roughly one striated tubule per three to four clathrin-coated vesicular profiles. However, whereas the clathrin-coated vesicular profiles are readily identified no matter the section plane, the striated tubules can be identified unequivocally only in longitudinal sections. Because many tubules will be cross-sectioned, we find it likely that there is close to one tubule per clathrin-coated pit or aggregate of pits. The number of clathrin-coated structures at the cell surface in CHC antisense RNA-expressing cells from 2 to 6 days of induction was quantified and found to be 3- to 5-fold higher than in control cells (Table 1). Similarly, the number of striated tubules appeared constant from 2 to 6 days of induction. Thus, CHC still present at this time is able to form coat structures, but the coated pits cannot pinch off. Whether these coated structures contain some partially cleaved molecules interfering with normal clathrin function is not known. The first signs of multiple coated pit cgi doi pnas Iversen et al.

5 Table 1. Expression of CHC antisense RNA increases the number of clathrin coats associated with the cell surface Growth conditions No. of clathrin-coated profiles per 100 m of cell surface No. of cell profiles examined tet, 48 h tet, 48 h tet, 96 h tet, 144 h Cells were grown with ( ) or without ( ) tet for the indicated times. Values shown are mean SD. tubular structures (Fig. 3 C E). Thus, in CHC antisense RNAexpressing cells, clathrin-coated pits accumulated at the cell surface, connected to the plasma membrane via long, dynaminwrapped necks. The reason for this phenotype is not obvious but could depend on, although it is not caused by, the increased expression of endogenous dynamin (see below). Another striking structural effect of antisense-chc expression was a highly pleiomorphic, multivesicular compartment (Fig. 4). This compartment was not associated with the cell surface, because it was not stained after fixation with ruthenium red (data not shown). In contrast, it contained internalized horseradish peroxidase (data not shown), and we therefore consider it a modified endosome compartment. This multivesicular compartment was associated with numerous 70- to 120-nm clathrin-coated vesicular structures (which were rarely observed on endosomes in control cells) (Fig. 4). This image is reminiscent of images observed in shibire nerve terminals during CELL BIOLOGY SEE COMMENTARY Fig. 3. (A and B) Representative examples of the peripheral cytoplasm of BHK21-tTA anti-chc cells expressing CHC antisense RNA (without tet for 2 days) and fixed in the presence of ruthenium red. Note the ruthenium redstained clathrin-coated profiles (arrowheads). One such profile is associated with a tubule (large arrow in A); several very small vesicular profiles, which may actually represent cross-sectioned tubules, are also shown (small arrows in A). (C E) Ultracryosections of the cells expressing CHC antisense RNA (for 3 days). The sections are ImmunoGold-labeled (with Hudy 1 followed by 5-nm gold particles) to identify dynamin, which is present not only on clathrincoated vesicular profiles (arrowheads) but also on tubular structures (arrows). (Bar 200 nm.) structures and some formation of striated tubules were noticed after 18 h of CHC antisense RNA induction, suggesting that their formation correlated with inhibition of endocytosis. The cross-striated 20- to 30-nm tubules connecting the clathrin-coated pits to the cell surface in CHC antisense RNAexpressing cells resemble the dynamin-wrapped necks of clathrin-coated structures found in permeabilized synaptosomes after incubation with guanosine 5 -[ -thio]triphosphate (22), or upon overexpression of mutant dynamin (8). Thus, we incubated ultracryosections of CHC antisense RNA-expressing cells with an anti-dynamin antibody (Hudy 1). This antibody not only labeled the clathrin coats as reported (16, 18), but also the Fig. 4. BHK21-tTA anti-chc cells expressing CHC antisense RNA (without tet for 4 days). Note the numerous clathrin-coated buds (arrowheads) on the pleiomorphic, multivesicular compartment. (Bar 200 nm.) Iversen et al. PNAS April 29, 2003 vol. 100 no

6 Fig. 5. Expression of dynamin is increased in the CHC antisense RNA cells. (A) Increased rate of dynamin synthesis. The cells were grown with or without tet for 2 days. Then they were pulse-labeled with [ 35 S]Met [ 35 S]Cys (30 min) and solubilized. Dynamin was immunoprecipitated and resolved on a 10% SDS PAGE gel. (B) Northern blot analysis of dynamin 2 mrna. Total cellular RNA was isolated from cells grown with or without tet for 2 days, and 10 and 20 g of each RNA sample, as indicated, were loaded onto a 1% agarose formaldehyde gel and blotted onto a nylon membrane. Hybridization was carried out at 60 C with a labeled cdna probe for dynamin 2. Band intensities on the blot were quantified with a PhosphorImager. recovery from a temperature block (23). Interestingly, in contrast to the situation at the plasma membrane, the endosomeassociated clathrin-coated pits never showed tubular neck or ring structures, even though a high level of dynamin was present in the cytosol (see below), suggesting differences in the mechanism of vesicle formation at the two donor membranes. Despite accumulation of clathrin on endosomes, endocytosed transferrin seemed to be exocytosed at a normal rate (data not shown). Dynamin Synthesis Is Increased upon CHC Antisense RNA Expression. The observation of accumulated clathrin-coated pits in the CHC antisense RNA cells made us investigate expression levels of different proteins participating in clathrin-coated vesicle formation (24). Western blot analysis revealed that the steady-state level of dynamin was increased 20-fold, whereas there was no change in the other proteins investigated (clathrin light chain, AP1, AP2, Eps15, epsin, amphiphysin, and endophilin; data not shown). Furthermore, the synthesis of dynamin was found to be up-regulated 10-fold as determined by a metabolic pulse experiment (Fig. 5A). This increase also was reflected in different Northern blot analysis experiments. An 5-fold higher level of dynamin 2 mrna in the CHC antisense RNA-expressing cells is demonstrated in Fig. 5B. Interestingly, the time course of this increased level of dynamin corresponded to the time course of the inhibition of transferrin endocytosis (data not shown), as if the cells tried to counteract the inhibition of clathrin-dependent endocytosis. Such an up-regulation might be required for formation of dynamin rings in the cells studied here, but it is clearly not the explanation for this phenotype. Immunofluorescence microscopy of the uninduced BHK21-tTA anti-chc cells transiently overexpressing hemagglutinin-tagged dynamin 2 wild type displayed no inhibition in uptake of Cy3-labeled transferrin (data not shown). This agrees with previous studies of wild-type dynamin overexpression (18). In conclusion, despite an increased level of dynamin in the cells expressing CHC antisense RNA, dynamin at the neck of clathrin-coated pits in our CHC antisense RNA cells is not sufficient for release of vesicles. The function of dynamin in fission might require a critical level of clathrin or recently synthesized intact clathrin. Interestingly, our data suggest a new and unexpected link between synthesis of CHC and dynamin. We thank Jorunn Jacobsen, Anne-Grethe Myrann, Ulla Hjortenberg, Mette Ohlsen, Keld Ottosen, and Kirsten Pedersen for expert technical assistance. This work was supported by the Norwegian and the Danish Cancer Societies, the Norwegian Research Council for Science and the Humanities, the Danish Medical Research Council, the Novo Nordisk Foundation, the Jahre Foundation, and the Jeanette and Søren Bothners Legacy. 1. Traub, L. M., Bannykh, S. I., Rodel, J. E., Aridor, M., Balch, W. E. & Kornfeld, S. (1996) J. Cell Biol. 135, Stoorvogel, W., Oorschot, V. & Geuze, H. J. (1996) J. Cell Biol. 132, Johannes, L. & Goud, B. (2000) Traffic 1, van Dam, E. M. & Stoorvogel, W. (2002) Mol. Biol. Cell 13, Raiborg, C., Bache, K. G., Gillooly, D. J., Madshus, I. H., Stang, E. & Stenmark, H. (2002) Nat. Cell Biol. 4, Sachse, M., Urbe, S., Oorschot, V., Strous, G. J. & Klumperman, J. (2002) Mol. Biol. Cell 13, Bennett, E. M., Lin, S. X., Towler, M. C., Maxfield, F. R. & Brodsky, F. M. (2001) Mol. Biol. Cell 12, Marks, B., Stowell, M. H., Vallis, Y., Mills, I. G., Gibson, A., Hopkins, C. R. & McMahon, H. T. (2001) Nature 410, Sandvig, K. & van Deurs, B. (2002) Annu. Rev. Cell Dev. Biol. 18, Lamaze, C., Dujeancourt, A., Baba, T., Lo, C. G., Benmerah, A. & Dautry- Varsat, A. (2001) Mol. Cell 7, Fraker, P. J. & Speck, J. C., Jr. (1978) Biochem. Biophys. Res. Commun. 80, Press, B., Feng, Y., Hoflack, B. & Wandinger-Ness, A. (1998) J. Cell Biol. 140, Kirchhausen, T., Harrison, S. C., Chow, E. P., Mattaliano, R. J., Ramachandran, K. L., Smart, J. & Brosius, J. (1987) Proc. Natl. Acad. Sci. USA 84, de la Luna, S., Soria, I., Pulido, D., Ortin, J. & Jimenez, A. (1988) Gene 62, Skretting, G., Torgersen, M. L., van Deurs, B. & Sandvig, K. (1999) J. Cell Sci. 112, Nicoziani, P., Vilhardt, F., Llorente, A., Hilout, L., Courtoy, P. J., Sandvig, K. & van Deurs, B. (2000) Mol. Biol. Cell 11, Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, Damke, H., Baba, T., Warnock, D. E. & Schmid, S. L. (1994) J. Cell Biol. 127, Acton, S. L. & Brodsky, F. M. (1990) J. Cell Biol. 111, Fenteany, G., Standaert, R. F., Lane, W. S., Choi, S., Corey, E. J. & Schreiber, S. L. (1995) Science 268, Geier, E., Pfeifer, G., Wilm, M., Lucchiari-Hartz, M., Baumeister, W., Eichmann, K. & Niedermann, G. (1999) Science 283, Takei, K., McPherson, P. S., Schmid, S. L. & De Camilli, P. (1995) Nature 374, Koenig, J. H. & Ikeda, K. (1989) J. Neurosci. 9, Takei, K. & Haucke, V. (2001) Trends Cell Biol. 11, Satin, B., Norais, N., Telford, J., Rappuoli, R., Murgia, M., Montecucco, C. & Papini, E. (1997) J. Biol. Chem. 272, cgi doi pnas Iversen et al.

SANTA CRUZ BIOTECHNOLOGY, INC.

SANTA CRUZ BIOTECHNOLOGY, INC. TECHNICAL SERVICE GUIDE: Western Blotting 2. What size bands were expected and what size bands were detected? 3. Was the blot blank or was a dark background or non-specific bands seen? 4. Did this same

More information

Technical Note Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit IRDye 680RD

Technical Note Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit IRDye 680RD Technical Note Detection of post-immunoprecipitation proteins by Western blot using the Quick Western Kit IRDye 680RD Developed for: Aerius, Odyssey Classic, Odyssey CLx and Odyssey Sa Imaging Systems

More information

Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde

Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde Supplementary text Supplementary materials and methods Histopathological examination Segments of the obstructed intestinal loops were fixed in 4% paraformaldehyde (PFA) and embedded in paraffin wax with

More information

This Document Contains:

This Document Contains: This Document Contains: 1. In-Cell Western Protocol II. Cell Seeding and Stimulation Supplemental Protocol III. Complete Assay Example: Detailing the Seeding, Stimulation and Detection of the A431 Cellular

More information

Supplemental Information. PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock

Supplemental Information. PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock Molecular Cell, Volume 49 Supplemental Information PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock Dafne Campigli Di Giammartino, Yongsheng Shi, and James L. Manley Supplemental Information

More information

Ral Activation Assay Kit

Ral Activation Assay Kit Product Manual Ral Activation Assay Kit Catalog Number STA-408 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Small GTP-binding proteins (or GTPases) are a family of

More information

Fisher (Fairlawn, NJ) and Sigma-Aldrich (St. Louis, MO) and were used without further. (Promega) and DpnI (New England Biolabs, Beverly, MA).

Fisher (Fairlawn, NJ) and Sigma-Aldrich (St. Louis, MO) and were used without further. (Promega) and DpnI (New England Biolabs, Beverly, MA). 175 Appendix III Chapter 4 Methods General. Unless otherwise noted, reagents were purchased from the commercial suppliers Fisher (Fairlawn, NJ) and Sigma-Aldrich (St. Louis, MO) and were used without further

More information

Protocol for induction of expression and cell lysate production

Protocol for induction of expression and cell lysate production Protocol for induction of expression and cell lysate production AV-04 Doxycyclin induction and cell lysate 1.0 Introduction / Description This method is intended for the treatment of the previously transfected

More information

Supplemental Online Material. The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/-

Supplemental Online Material. The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/- #1074683s 1 Supplemental Online Material Materials and Methods Cell lines and tissue culture The mouse embryonic fibroblast cell line #10 derived from β-arrestin1 -/- -β-arrestin2 -/- knock-out animals

More information

The preparation of native chromatin from cultured human cells.

The preparation of native chromatin from cultured human cells. Native chromatin immunoprecipitation protocol The preparation of native chromatin from cultured human cells. All solutions need to be ice cold. Sucrose containing solutions must be made up fresh on the

More information

WesternMAX Alkaline Phosphatase Chemiluminescent Detection Kits

WesternMAX Alkaline Phosphatase Chemiluminescent Detection Kits WesternMAX Alkaline Phosphatase Chemiluminescent Detection Kits Code N221-KIT N220-KIT Description WesternMAX Chemiluminescent AP Kit, Anti-Mouse Includes: Alkaline Phosphatase (AP) Conjugated Anti-Mouse

More information

OPPF-UK Standard Protocols: Mammalian Expression

OPPF-UK Standard Protocols: Mammalian Expression OPPF-UK Standard Protocols: Mammalian Expression Joanne Nettleship joanne@strubi.ox.ac.uk Table of Contents 1. Materials... 3 2. Cell Maintenance... 4 3. 24-Well Transient Expression Screen... 5 4. DNA

More information

In-Cell Western Kits I and II

In-Cell Western Kits I and II Odyssey and Aerius Infrared Imaging Systems In-Cell Western Assay Kits I and II Published November, 2006. The most recent version of this protocol is posted at http://biosupport.licor.com/protocols.jsp

More information

Rho activation kit. Catalog Number: ADI-EKS-465. Table of Contents

Rho activation kit. Catalog Number: ADI-EKS-465. Table of Contents Rho activation kit Catalog Number: ADI-EKS-465 Table of Contents Assay Design Page 1 Scientific Overview 2 Precautions 2 Materials Provided 3 Storage of Materials 3 Materials Required but Not Provided

More information

IMMUNOPRECIPITATION (IP)

IMMUNOPRECIPITATION (IP) 1 IMMUNOPRECIPITATION (IP) Overview and Technical Tips 2 CONTENTS 3 7 8 9 12 13 17 18 19 20 Introduction Factors Influencing IP General Protocol Modifications Of IP Protocols Troubleshooting Contact Us

More information

Dolphin-Chemi Plus. Aim: To visualise and evaluate the performance of chemiluminescent immunoblots using Wealtec s Dolphin-Chemi plus image system

Dolphin-Chemi Plus. Aim: To visualise and evaluate the performance of chemiluminescent immunoblots using Wealtec s Dolphin-Chemi plus image system Application Note 03 Dolphin-Chemi plus 8/22/2007 Dolphin-Chemi Plus Aim: To visualise and evaluate the performance of chemiluminescent immunoblots using Wealtec s Dolphin-Chemi plus image system INTRODUCTION

More information

western blotting tech

western blotting tech western blotting tech note 6148 Transfer of High Molecular Weight Proteins to Membranes: A Comparison of Transfer Efficiency Between Blotting Systems Nik Chmiel, Bio-Rad Laboratories, Inc., 6000 James

More information

sirna Transfection Into Primary Neurons Using Fuse-It-siRNA

sirna Transfection Into Primary Neurons Using Fuse-It-siRNA sirna Transfection Into Primary Neurons Using Fuse-It-siRNA This Application Note describes a protocol for sirna transfection into sensitive, primary cortical neurons using Fuse-It-siRNA. This innovative

More information

Supplemental Data. LMO4 Controls the Balance between Excitatory. and Inhibitory Spinal V2 Interneurons

Supplemental Data. LMO4 Controls the Balance between Excitatory. and Inhibitory Spinal V2 Interneurons Neuron, Volume 61 Supplemental Data LMO4 Controls the Balance between Excitatory and Inhibitory Spinal V2 Interneurons Kaumudi Joshi, Seunghee Lee, Bora Lee, Jae W. Lee, and Soo-Kyung Lee Supplemental

More information

MEK1/2 (MAPK Kinase) Activity Assay Kit

MEK1/2 (MAPK Kinase) Activity Assay Kit MEK1/2 (MAPK Kinase) Activity Assay Kit For 96 tests Cat. No. SGT440 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA & Canada Phone: +1(800) 437-7500 Fax: +1 (951) 676-9209 Europe +44 (0)

More information

Myers Lab ChIP-seq Protocol v Modified January 10, 2014

Myers Lab ChIP-seq Protocol v Modified January 10, 2014 Myers Lab ChIP-seq Protocol V011014 1 Contact information: Dr. Florencia Pauli Behn HudsonAlpha Institute for Biotechnology 601 Genome Way Huntsville, AL 35806 Telephone: 256-327-5229 Email: fpauli@hudsonalpha.org

More information

Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna

Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna Supplemental Materials and Methods Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna (Mission shrna, Sigma) against mouse MMP14 were transfected into HEK293 cells using FuGene6

More information

SensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric*

SensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric* Catalog # Kit Size SensoLyte Anti-alpha-Synuclein Quantitative ELISA Kit (Human/Mouse/Rat) *Colorimetric* AS-55550 One 96-well strip plate This kit is optimized to detect human/mouse/rat alpha-synuclein

More information

Arf6 Activation Assay Kit

Arf6 Activation Assay Kit Product Manual Arf6 Activation Assay Kit Catalog Number STA- 407-6 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Small GTP-binding proteins (or GTPases) are a family

More information

TECHNICAL BULLETIN. MEK Activity Assay Kit. Product Code CS0490 Storage Temperature 20 C

TECHNICAL BULLETIN. MEK Activity Assay Kit. Product Code CS0490 Storage Temperature 20 C MEK Activity Assay Kit Product Code CS0490 Storage Temperature 20 C TECHNICAL BULLETIN Product Description The MAP kinase kinases (MAPKK, mitogen-activated protein kinase kinase, also termed MEK) are a

More information

Strep-tag detection in Western blots

Strep-tag detection in Western blots Strep-tag detection in Western blots General protocol for the detection of Strep-tag fusion proteins Last date of revision April 2012 Version PR07-0010 www.strep-tag.com For research use only Important

More information

Data Sheet. PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit Catalog # Size: 96 reactions

Data Sheet. PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit Catalog # Size: 96 reactions Data Sheet PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit Catalog # 72003 Size: 96 reactions DESCRIPTION: Cell signaling through the PD-1 receptor upon binding the PD-L1 ligand attenuates immune

More information

Viral RNAi suppressor reversibly binds sirna to. outcompete Dicer and RISC via multiple-turnover

Viral RNAi suppressor reversibly binds sirna to. outcompete Dicer and RISC via multiple-turnover Supplementary Data Viral RNAi suppressor reversibly binds sirna to outcompete Dicer and RISC via multiple-turnover Renata A. Rawlings 1,2, Vishalakshi Krishnan 2 and Nils G. Walter 2 * 1 Biophysics and

More information

A General Protocol for GST Pull-down Lili Jing *

A General Protocol for GST Pull-down Lili Jing * A General Protocol for GST Pull-down Lili Jing * Department of Cell and Molecular Biology, University of Pennsylvania, Philadelphia, USA *For correspondence: lilijingcn@gmail.com [Abstract] GST pull-down

More information

Supporting Online Material for

Supporting Online Material for www.sciencemag.org/cgi/content/full/323/5910/124/dc1 Supporting Online Material for Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy Qian Yang, Hua She, Marla Gearing, Emanuela

More information

*Corresponding author. Tel: ;

*Corresponding author. Tel: ; 1 SUPPLEMENTARY DATA 2 3 4 5 6 7 8 9 10 11 Integrin 2 1 in nonactivated conformation can induce focal adhesion kinase signaling Maria Salmela 1, Johanna Jokinen 1,2, Silja Tiitta 1, Pekka Rappu 1, Holland

More information

KPL SignaLOCK ChemiWestern Kits (Film and Imager Analysis)

KPL SignaLOCK ChemiWestern Kits (Film and Imager Analysis) KPL SignaLOCK ChemiWestern Kits (Film and Imager Analysis) SignaLOCK HRP ChemiWestern Kit (Film) Catalog No. 54-53-00 SignaLOCK HRP ChemiWestern Kit (Imager) Catalog No. 54-54-00 SignaLOCK AP ChemiWestern

More information

IMMUNOPRECIPITATION TROUBLESHOOTING TIPS

IMMUNOPRECIPITATION TROUBLESHOOTING TIPS IMMUNOPRECIPITATION TROUBLESHOOTING TIPS Creative Diagnostics Abstract Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds

More information

MATERIAL DATA SHEET. NOTE: Kit contains reagents sufficient for 10 x 30 μl reactions and 5 Western Blots (minigel. Reagents Provided in Kit

MATERIAL DATA SHEET. NOTE: Kit contains reagents sufficient for 10 x 30 μl reactions and 5 Western Blots (minigel. Reagents Provided in Kit Lot # XXXXX ITCH/AIP4 Ubiquitin Ligase Kit Cat. # K-270 MATERIAL DATA SHEET The mammalian Itchy homolog, or ITCH, (also known as Atrophin-1-interacting protein 4 or AIP4) is a HECT domain class ubiquitin

More information

Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit

Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit Application Note 13 RNA Sample Preparation Non-Organic-Based Isolation of Mammalian microrna using Norgen s microrna Purification Kit B. Lam, PhD 1, P. Roberts, MSc 1 Y. Haj-Ahmad, M.Sc., Ph.D 1,2 1 Norgen

More information

Ras activation kit. Catalog Number: ADI-EKS-460. Table of Contents

Ras activation kit. Catalog Number: ADI-EKS-460. Table of Contents Ras activation kit Catalog Number: ADI-EKS-460 Table of Contents Assay Design Page 1 Scientific Overview 1 Precautions 1 Materials Provided 2 Storage of Materials 2 Materials Required but Not Provided

More information

Western BLoT Ultra Sensitive HRP Substrate

Western BLoT Ultra Sensitive HRP Substrate Cat. # T7104A For Research Use Western BLoT Ultra Sensitive Product Manual Table of Contents I. Description... 3 II. Components... 3 III. Storage... 3 IV. Materials Required but Not Provided... 3 V. Precautions...

More information

Orexin A (HUMAN, MOUSE, RAT, PORCINE, OVINE,

Orexin A (HUMAN, MOUSE, RAT, PORCINE, OVINE, Orexin A (HUMAN, MOUSE, RAT, PORCINE, OVINE, BOVINE) Western Blot Kit Protocol (Catalog #WBK-003-30) PHOENIX PHARMACEUTICALS, INC. TABLE OF CONTENTS 1. Kit Contents...2 2. Storage...2 3. Introduction...3

More information

KPL LumiGLO Reserve Chemiluminescent Substrate

KPL LumiGLO Reserve Chemiluminescent Substrate DESCRIPTION KPL LumiGLO Reserve contains a luminol-based chemiluminescent substrate designed for use with peroxidase-labeled (HRP) reporter molecules. KPL LumiGLO Reserve offers improvements in the way

More information

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Legends for Supplementary Tables. Supplementary Table 1. An excel file containing primary screen data. Worksheet 1, Normalized quantification data from a duplicated screen: valid

More information

Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay) Xin Zhang and Qingsong Liu *

Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay) Xin Zhang and Qingsong Liu * Autophagy Assays (LC3B immunofluorescence, LC3B western blot, acridine orange assay) Xin Zhang and Qingsong Liu * High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui, China *For correspondence:

More information

Cellular Fractionation

Cellular Fractionation Cellular Fractionation Lamond Lab Protocol 2007 More detailed protocol can be found here: http://www.lamondlab.com/f7nucleolarprotocol.htm This protocol has been adapted to fractionate a variety of different

More information

Mitochondrial DNA Isolation Kit

Mitochondrial DNA Isolation Kit Mitochondrial DNA Isolation Kit Catalog Number KA0895 50 assays Version: 03 Intended for research use only www.abnova.com Table of Contents Introduction... 3 Background... 3 General Information... 4 Materials

More information

PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%)

PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%) 1 AFFINITY HIS-TAG PURIFICATION PROCEDURE FOR USE NICKEL NTA Magnetic Agarose Beads (5%) DESCRIPTION Nickel NTA Magnetic Agarose Beads are products that allow rapid and easy small-scale purification of

More information

MSD Immuno-Dot-Blot Assays. A division of Meso Scale Diagnostics, LLC.

MSD Immuno-Dot-Blot Assays. A division of Meso Scale Diagnostics, LLC. MSD Immuno-Dot-Blot Assays Example: High Throughput Western Blots Replacements Traditional Western Blots High content Molecular weight and immunoreactivity Labor and protein intensive Inherently low throughput

More information

Figure 1. Schematic of Ats1p expression plasmid.

Figure 1. Schematic of Ats1p expression plasmid. Abstract: Anita Corbett page 2 The goal of my rotation project was to express, purify, and examine the exchange activity of a putative guanine nucleotide exchange factor, Ats1p. The S. cerevisiae ATS1

More information

Cytoplasmic & Nuclear RNA Purification Kit Product # 21000, 37400

Cytoplasmic & Nuclear RNA Purification Kit Product # 21000, 37400 3430 Schmon Parkway Thorold, ON, Canada L2V 4Y6 Phone: 866-667-4362 (905) 227-8848 Fax: (905) 227-1061 Email: techsupport@norgenbiotek.com Cytoplasmic & Nuclear RNA Purification Kit Product # 21000, 37400

More information

Blot: a spot or stain, especially of ink on paper.

Blot: a spot or stain, especially of ink on paper. Blotting technique Blot: a spot or stain, especially of ink on paper. 2/27 In molecular biology and genetics, a blot is a method of transferring proteins, DNA or RNA, onto a carrier (for example, a nitrocellulose,pvdf

More information

Positively Charged Membrane

Positively Charged Membrane BIOBOND NYLON MEMBRANES ProductInformation Technical Bulletin No. MB-570 June 1999 Size Quantity Positively Charged Membrane Neutral Membrane 30 cm x 3.5 m 1 roll N4781 N1031 30 cm x 12 m 1 roll N4906

More information

EGFR (Phospho-Ser695)

EGFR (Phospho-Ser695) Assay Biotechnology Company www.assaybiotech.com Tel: 1-877-883-7988 Fax: 1-877-610-9758 EGFR (Phospho-Ser695) Colorimetric Cell-Based ELISA Kit Catalog #: OKAG02090 Please read the provided manual entirely

More information

Kinase Reaction and Alkylation Protocol

Kinase Reaction and Alkylation Protocol Kinase Reaction and Alkylation Protocol Protocol for the treatment of substrates prior to detection by Thiophosphate Ester antibodies This product is for research use only and is not intended for diagnostic

More information

Biotin 3' End DNA Labeling Kit

Biotin 3' End DNA Labeling Kit INSTRUCTIONS Biotin 3' End DNA Labeling Kit 3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105 89818 1290.4 Number Description 89818 Biotin 3' End DNA Labeling Kit, sufficient reagents to perform 20

More information

Western Blotting Detection Reagents

Western Blotting Detection Reagents Electrophoresis Western Blotting Detection Reagents Maximize Western Blot Detection Solutions for Any Blotting Application Choose the Best Approach for Your Needs When it comes to western blot detection,

More information

Cdc42 Activation Assay Kit

Cdc42 Activation Assay Kit Product Manual Cdc42 Activation Assay Kit Catalog Number STA- 402 20 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Small GTP-binding proteins (or GTPases) are a family

More information

INOS. Colorimetric Cell-Based ELISA Kit. Catalog #: OKAG00807

INOS. Colorimetric Cell-Based ELISA Kit. Catalog #: OKAG00807 INOS Colorimetric Cell-Based ELISA Kit Catalog #: OKAG00807 Please read the provided manual entirely prior to use as suggested experimental protocols may have changed. Research Purposes Only. Not Intended

More information

96-well Checkpoint Kinase Activity Assay Kit

96-well Checkpoint Kinase Activity Assay Kit Product Manual 96-well Checkpoint Kinase Activity Assay Kit Catalog Number STA-414 STA-414-5 96 assays 5 x 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Cdc25C is a

More information

Experimental Protocol for Multiplex Fluorescent Blotting Using the ChemiDoc MP Imaging System

Experimental Protocol for Multiplex Fluorescent Blotting Using the ChemiDoc MP Imaging System Experimental Protocol for Multiplex Fluorescent Blotting Using the ChemiDoc MP Imaging System Protocol Bulletin 6570 Stefanie L. Ritter and Donald G. Rainie, Deparment of Behavioral Neuroscience and Psychiatric

More information

NOTE ACRYLAMIDE IS NEUROTOXIN YOU MUST WEAR GLOVES.

NOTE ACRYLAMIDE IS NEUROTOXIN YOU MUST WEAR GLOVES. GST Purfication and Pulldown Part I Instructor: David Deitcher TA: Kristy Lawton In order to study the function of a protein it is often useful to have that protein purified away from others in the cell.

More information

jetpei In vitro Transfection Protocol

jetpei In vitro Transfection Protocol jetpei Cationic polymer transfection reagent In vitro Transfection Protocol 101-05 0.5 ml (250 transfections in 24-well plates) 101-05N 0.5 ml 50ml of 150 mm NaCl (250 transfections in 24-well plates)

More information

TECHNICAL BULLETIN NUCLEI EZ PREP NUCLEI ISOLATION KIT. Product Number NUC-101 Store at 2-8 C

TECHNICAL BULLETIN NUCLEI EZ PREP NUCLEI ISOLATION KIT. Product Number NUC-101 Store at 2-8 C NUCLEI EZ PREP NUCLEI ISOLATION KIT Product Number NUC-101 Store at 2-8 C TECHNICAL BULLETIN Product Description Sigma s Nuclei EZ Prep Kit is designed for the rapid isolation of nuclei from mammalian

More information

O-GlcNAcase Activity Assay

O-GlcNAcase Activity Assay O-GlcNAcase Activity Assay Prepared by Jen Groves and Junfeng Ma, The Johns Hopkins Unviersity School of Medicine Based on: Macauley MS et al., 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic

More information

TECHNICAL BULLETIN. GenElute mrna Miniprep Kit. Catalog MRN 10 MRN 70

TECHNICAL BULLETIN. GenElute mrna Miniprep Kit. Catalog MRN 10 MRN 70 GenElute mrna Miniprep Kit Catalog Numbers MRN 10, MRN 70 TECHNICAL BULLETIN Product Description The GenElute mrna Miniprep Kit provides a simple and convenient way to purify polyadenylated mrna from previously

More information

2.5. Equipment and materials supplied by user PCR based template preparation Influence of temperature on in vitro EGFP synthesis 11

2.5. Equipment and materials supplied by user PCR based template preparation Influence of temperature on in vitro EGFP synthesis 11 Manual 15 Reactions LEXSY in vitro Translation Cell-free protein expression kit based on Leishmania tarentolae for PCR-based template generation Cat. No. EGE-2010-15 FOR RESEARCH USE ONLY. NOT INTENDED

More information

Rotation Report Sample Version 2. Due Date: August 9, Analysis of the Guanine Nucleotide Exchange Activity of the S. cerevisiae Ats1 Protein

Rotation Report Sample Version 2. Due Date: August 9, Analysis of the Guanine Nucleotide Exchange Activity of the S. cerevisiae Ats1 Protein Rotation Report Sample Version 2 Due Date: August 9, 1998 Analysis of the Guanine Nucleotide Exchange Activity of the S. cerevisiae Ats1 Protein Anita H. Corbett Advisor: Amy Jones Rotation 1 Abstract:

More information

MANUALL TUBEs: Tandem Ubiquitin Binding Entities. Biotinylated K63-TUBE 1. Catalog Numbers: UM304

MANUALL TUBEs: Tandem Ubiquitin Binding Entities. Biotinylated K63-TUBE 1. Catalog Numbers: UM304 - 1 - MANUALL Biotinylated K63-TUBE 1 Catalog Numbers: UM304 all products are for research use only not intended for human or animal diagnostic or therapeutic uses LifeSensors, Inc., 271 Great Valley Parkway,

More information

His- Tag Protein ELISA Kit

His- Tag Protein ELISA Kit Revised Protocol Product Manual His- Tag Protein ELISA Kit Catalog Numbers AKR- 130 96 wells FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction A polyhistidine-tag, or His-tag, is

More information

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double mutant mice Belal A. Mohamed, Amal Z. Barakat, Torsten Held, Manar Elkenani, Christian Mühlfeld, Jörg Männer, and Ibrahim M. Adham

More information

Proteome Profiler TM 96

Proteome Profiler TM 96 Proteome Profiler TM 96 Mouse Phospho-RTK Custom Array Catalog Number ARZC03 For the parallel determination of the relative levels of tyrosine phosphorylation of mouse receptor tyrosine kinases (RTKs).

More information

MMLV Reverse Transcriptase 1st-Strand cdna Synthesis Kit

MMLV Reverse Transcriptase 1st-Strand cdna Synthesis Kit MMLV Reverse Transcriptase 1st-Strand cdna Synthesis Kit Cat. No. MM070150 Available exclusively thru Lucigen. lucigen.com/epibio www.lucigen.com MA265E MMLV Reverse Transcriptase 1st-Strand cdna Synthesis

More information

NAG-1 (GDF-15, MIC-1) Prostate Cancer ELISA kit

NAG-1 (GDF-15, MIC-1) Prostate Cancer ELISA kit NAG-1 (GDF-15, MIC-1) Prostate Cancer ELISA kit Catalog Number: NG1 Store at -0 C. FOR RESEARCH USE ONLY v. 1081 Introduction This sandwich ELISA kit is for determination of NAG-1 (GDF-15, MIC-1) levels

More information

WesternBright Quantum

WesternBright Quantum User Manual WesternBright Quantum Chemiluminescent HRP Substrate For Catalog Numbers K-12042-C20 20 ml, sufficient for 200 cm 2 K-12042-D10 100 ml, sufficient for 1000 cm 2 K-12042-D20 200 ml, sufficient

More information

ab Ubiquitylation Assay Kit

ab Ubiquitylation Assay Kit ab139467 Ubiquitylation Assay Kit Instructions for Use For the activation of ubiquitin for use in ubiquitylation experiments This product is for research use only and is not intended for diagnostic use.

More information

Azure Biosystems Western Blotting Workflow

Azure Biosystems Western Blotting Workflow Azure Biosystems Western Blotting Workflow PROBE PLAN SEPARATE ANALYZE VISUALIZE PLAN Plan your experiment and choose your detection method Chemiluminescent Western Blotting The most common method for

More information

E.Z.N.A. Tissue RNA Kit. R preps R preps

E.Z.N.A. Tissue RNA Kit. R preps R preps E.Z.N.A. Tissue RNA Kit R6688-00 5 preps R6688-01 50 preps May 2015 E.Z.N.A. Tissue RNA Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Important Notes...4 Homogenization

More information

SuperSignal West Pico Chemiluminescent Substrate

SuperSignal West Pico Chemiluminescent Substrate INSTRUCTIONS SuperSignal West Pico Chemiluminescent Substrate 3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105 34077 34078 34079 34080 0636.3 Number Description 34079 SuperSignal West Pico Chemiluminescent

More information

Note: for laboratory research use only. RNA High-purity Total RNA Rapid Extraction Kit (Spin-column) Signalway Biotechnology

Note: for laboratory research use only. RNA High-purity Total RNA Rapid Extraction Kit (Spin-column) Signalway Biotechnology Note: for laboratory research use only RNA High-purity Total RNA Rapid Extraction Kit (Spin-column) Cat. #: RP1202 (50preps) Signalway Biotechnology I. Kit Content, Storage Condition and Stability Content

More information

EXPIRED. The original method was described by F. Lasne et al. in Analytical Biochemistry 311 (2002)

EXPIRED. The original method was described by F. Lasne et al. in Analytical Biochemistry 311 (2002) HARMONIZATION OF THE METHOD FOR THE IDENTIFICATION OF EPOETIN ALFA AND BETA (EPO) AND DARBEPOETIN ALFA (NESP) BY IEF-DOUBLE BLOTTING AND CHEMILUMINESCENT DETECTION. The criteria presented herein have been

More information

Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila

Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila Cell Supplemental Information Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila Bo Liu, Yonggang Zheng, Feng Yin, Jianzhong Yu, Neal Silverman, and Duojia Pan Supplemental Experimental

More information

EPIGENTEK. EpiQuik In Situ Histone H3-K27 Tri-Methylation Assay Kit. Base Catalog # P-3014T PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE

EPIGENTEK. EpiQuik In Situ Histone H3-K27 Tri-Methylation Assay Kit. Base Catalog # P-3014T PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE EpiQuik In Situ Histone H3-K27 Tri-Methylation Assay Kit Base Catalog # PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The EpiQuik In Situ Histone H3-K27 Tri-Methylation Assay Kit is suitable for specifically

More information

Supporting Information

Supporting Information Supporting Information Wiley-VCH 2006 69451 Weinheim, Germany Rolling-circle Amplification of a DNA Nanojunction Chenxiang Lin, Mingyi Xie, Julian J.L. Chen, Yan Liu and Hao Yan A. RCA replication of the

More information

SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes

SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes WHITE PAPER SuperScript IV Reverse Transcriptase SuperScript IV Reverse Transcriptase as a better alternative to AMV-based enzymes Abstract Reverse transcriptases (RTs) from avian myeloblastosis virus

More information

MagSi Beads. Magnetic Silica Beads for Life Science and Biotechnology study

MagSi Beads. Magnetic Silica Beads for Life Science and Biotechnology study MagSi Beads Magnetic Silica Beads for Life Science and Biotechnology study MagnaMedics Diagnostics B.V. / Rev. 9.2 / 2012 Wide range of products for numerous applications MagnaMedics separation solutions

More information

Supporting Online Material for

Supporting Online Material for www.sciencemag.org/cgi/content/full/1137999/dc1 Supporting Online Material for Disrupting the Pairing Between let-7 and Enhances Oncogenic Transformation Christine Mayr, Michael T. Hemann, David P. Bartel*

More information

Supplementary information for. An Ultrasensitive Biosensor for DNA Detection Based on. Hybridization Chain Reaction Coupled with the Efficient

Supplementary information for. An Ultrasensitive Biosensor for DNA Detection Based on. Hybridization Chain Reaction Coupled with the Efficient Supplementary information for An Ultrasensitive Biosensor for DNA Detection Based on Hybridization Chain Reaction Coupled with the Efficient Quenching of Ruthenium Complex to CdTe Quantum Dot Yufei Liu,

More information

Strep-tag Technology for Molecular Weight (MW) Determinations on Blots Using Precision Plus Protein Standards

Strep-tag Technology for Molecular Weight (MW) Determinations on Blots Using Precision Plus Protein Standards blotting tech note 2847 Strep-tag Technology for Molecular Weight (MW) Determinations on Blots Using Precision Plus Protein Standards Introduction Bio-Rad has consistently provided innovative standards

More information

TECHNICAL BULLETIN. JNK 1&2 Activity Assay Kit. Product Number CS0380 Storage Temperature 20 C

TECHNICAL BULLETIN. JNK 1&2 Activity Assay Kit. Product Number CS0380 Storage Temperature 20 C JNK 1&2 Activity Assay Kit Product Number CS0380 Storage Temperature 20 C TECHNICAL BULLETIN Product Description The c-jun N-terminal kinases (JNKs), also known as stress activated protein kinases (SAPKs),

More information

Gene Expression Technology

Gene Expression Technology Gene Expression Technology Bing Zhang Department of Biomedical Informatics Vanderbilt University bing.zhang@vanderbilt.edu Gene expression Gene expression is the process by which information from a gene

More information

Human immunoglobulin G(IgG) ELISA Kit

Human immunoglobulin G(IgG) ELISA Kit Human immunoglobulin G(IgG) ELISA Kit For the quantitative determination of human immunoglobulin G (IgG) concentrations in serum, plasma, cell culture supernates, urine, tissue homogenates, cell lysates.

More information

Human TGF-beta1 ELISA

Human TGF-beta1 ELISA K-ASSAY Human TGF-beta1 ELISA For the quantitative determination of TGF-beta1 in human cell culture supernates, serum, plasma (EDTA) and urine Cat. No. KT-1471 For Research Use Only. Not for diagnostic

More information

Western Blotting Products. A Complete Source for All Your Blotting Needs

Western Blotting Products. A Complete Source for All Your Blotting Needs Western Blotting Products A Complete Source for All Your Blotting Needs A History of Innovation and Leadership A pioneer of western blotting apparatus, Bio-Rad has come to be considered the industry leader

More information

In-Gel Western Detection Using Near-Infrared Fluorescence

In-Gel Western Detection Using Near-Infrared Fluorescence In-Gel Western Detection Using Near-Infrared Fluorescence Developed for: Aerius, and Odyssey Family of Imagers Please refer to your manual to confirm that this protocol is appropriate for the applications

More information

MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr.

MIT Department of Biology 7.013: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. MIT Department of Biology 7.01: Introductory Biology - Spring 2005 Instructors: Professor Hazel Sive, Professor Tyler Jacks, Dr. Claudette Gardel iv) Would Xba I be useful for cloning? Why or why not?

More information

Tyrosine Kinase Assay Kit

Tyrosine Kinase Assay Kit Instruction Manual for Tyrosine Kinase Assay Kit Colorimetric Detection Catalog # 17-315 Direct ELISA system for the colorimetric detection of protein tyrosine phosphotransferase activity using a biotinylated

More information

Lecture Four. Molecular Approaches I: Nucleic Acids

Lecture Four. Molecular Approaches I: Nucleic Acids Lecture Four. Molecular Approaches I: Nucleic Acids I. Recombinant DNA and Gene Cloning Recombinant DNA is DNA that has been created artificially. DNA from two or more sources is incorporated into a single

More information

XactEdit Cas9 Nuclease with NLS User Manual

XactEdit Cas9 Nuclease with NLS User Manual XactEdit Cas9 Nuclease with NLS User Manual An RNA-guided recombinant endonuclease for efficient targeted DNA cleavage Catalog Numbers CE1000-50K, CE1000-50, CE1000-250, CE1001-250, CE1001-1000 Table of

More information

Binding and Phosphorylation of Tubulin by G Protein-coupled Receptor Kinases*

Binding and Phosphorylation of Tubulin by G Protein-coupled Receptor Kinases* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 32, Issue of August 7, pp. 20308 20316, 1998 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Binding and Phosphorylation

More information

Discovery and Humanization of Novel High Affinity Neutralizing Monoclonal Antibodies to Human IL-17A

Discovery and Humanization of Novel High Affinity Neutralizing Monoclonal Antibodies to Human IL-17A Discovery and Humanization of Novel High Affinity Neutralizing Monoclonal Antibodies to Human IL-17A Contacts: Marty Simonetti martysimonetti@gmail.com Kirby Alton kirby.alton@abeomecorp.com Rick Shimkets

More information

E.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps

E.Z.N.A. Yeast Plasmid Mini Kit. D preps D preps E.Z.N.A. Yeast Plasmid Mini Kit D3376-00 5 preps D3376-01 50 preps November 2015 E.Z.N.A. Yeast Plasmid Mini Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Preparing

More information

Transferrin Conjugates

Transferrin Conjugates Transferrin Conjugates Table 1 Contents and storage Material Formulation Storage Stability Transferrin conjugates* lyophilized powder containing transferrin conjugate, lyophilized in phosphate-buffered

More information

Tropix Chemiluminescent Kits and Reagents For Cell Biology Applications

Tropix Chemiluminescent Kits and Reagents For Cell Biology Applications PRODUCT FAMILY BULLETIN Tropix Chemiluminescent Kits and Reagents Tropix Chemiluminescent Kits and Reagents For Cell Biology Applications Introduction to Chemiluminescence Chemiluminescence is the conversion

More information