Journal of Cell Science Supplementary Material
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4 Figure S4 A His-(RBCPS)T15 His-(_BCPS) T15 His-PXN His-PXN+T15 His-PXN+ (RB_PS)T15 Myc-Ub IP:Ni-NTA 6M GuHCL WB: anti-myc WB: anti-his (Ub)n His-PXN His-(RBCPS)T15 His-(_BCPS)T kda -75 kda -50 kda -100 kda -75 kda -50 kda B % Open wound area (72h) NTsi * T15si YFP YFP RBCPS _BCPS T15-YFP (si-res) *
5 Online supplemental material Supplementary figure and Movie legends Fig. S1. The coiled coil domain of TRIM15 is essential for targeting to focal adhesions. (A) Columns 1-4 show split and merged images of HeLa cells expressing YFP-tagged (green) zyxin, paxillin, talin or actin and CFP-tagged or immunolocalized untagged TRIM15 (anti-trim15; red). The arrows in the third column indicate cytoplasmic bodies. Scale bar: 10 µm. (B) Localization of Human (Hs) or mouse (Mm) YFP-tagged TRIM15 in indicated cell types. Scale bar: 10 μm. Fig. S2. The oligomerization activity of B-box contributes to interaction with paxillin in addition to coiled coil domain of TRIM15 that harbors the paxillin binding site. (A & B) Sequence alignment of a stretch in TRIM15 coiled coil region (aa ) with the putative paxillin binding subdomain (PBS) of vinculin, actopaxin and FAK and with TRIM15 protein from hsa (Homo sapiens; human), ptr (Pan troglodytes; chimpanzee), mcc (Macaca mulatta; rhesus macaques), bta (Bos taurus; cow) and mmu (Mus musculus; mice) showing the conserved putative paxillin binding subdomain. (C) A secondary structure analyses predicted using the PSPIRED v3.3 software (Jones, 1999) for TRIM15 amino acid sequence ( ) encompassing the coiled coil domain is shown along with confidence of prediction (blue). The cylindrical regions shown in pink depict the predicted helical regions [H] and the lines represent the loops [L]. The amino acids corresponding to putative PBS (VTRLG) near the end of the coiled coil helical region is underlined and asterisks are shown below the mutated amino acids. (D) The ability of mutant YFP-tagged TRIM15 to dimerize was tested by co-expression with FLAG-tagged wild-type TRIM15 in HEK293 cells. Cell lysates were immunoprecipitated using FLAG antibodies followed by western blotting with antibodies to FLAG and GFP. (E) Gel filtration profile of wild-type and B-box deleted TRIM15 purified from E. coli. Fraction eluted at ~8 ml represents higher-order oligomers while fraction eluted at ~12 ml corresponds to a molecular weight of ~114 kda. The E. coli purified TRIM15 (RBCPS) and B-box deleted TRIM15 (R_CPS) are shown as coomassie stained gel along with molecular weight markers. Fig. S3. (A) Point mutations in coiled coil and B-box domain abolish focal adhesion targeting of TRIM15. TIRFM images of HeLa cells expressing indicated TRIM15-YFP mutants (green) from Fig. 1
6 B and the YFP fusion of the B-box domain of TRIM15 is shown here with talin-cfp as a focal adhesion reference marker to ensure that samples were imaged at the correct focal plane. This reference focal plane was critical to ascertain cytoplasmic fluorescence of TRIM15 mutants or individual domains that fail to localize to FAs. Individual FAs are outlined for clarity.scale bar: 10 μm. (B) TRIM15-depletion impairs cell migration and directional persistence in HT1080 cells. HT1080 cell monolayers treated with indicated sirnas with or without ectopic expression of sirnaresistant TRIM15 (T15si rescue) for 72 h were wounded and cell motility followed by live cell imaging for h. Selected frames of Movie S1 at 0 and 13 h after wounding are shown. Migration trajectories of individual cells are shown as dots and lines The outlines of representative cell boundaries and direction of migration are shown in the right to emphasize the single leading edge (shown in red) seen for NTsi- and T15si-treated rescued cells compared to multiple lateral lamellopodia in T15si-treated cells. (C-F) Plots of mentioned cell migration parameters for three experiments as in E are shown. The parameters for more than individual cells (open and closed circles) were computed. Statistical significance values ****, * correspond to p< and p<0.05, respectively. ns: not significant. Fig. S4. TRIM15 is a self-ubiquitinating E3 ubiquitin ligase but does not ubiquitinate paxillin and E3 activity is not required for its function. (A)TRIM15 self- and paxillin-ubiquitination were tested by expressing wild-type and RING-deleted His-tagged TRIM15 or paxillin in the presence of myc-tagged ubiquitin. His-tagged TRIM15 or paxillin were isolated using Ni-NTA under denaturing conditions using 6 M Guanidinium HCL and the ubiquitination tested by western blotting with antimyc antibodies. (B) Percent open wound area at 72 h determined using images analyzed by Tscratch software during the course of wound healing assay carried out in HeLa cells treated with either nontargeting (NT) or TRIM15 (T15) sirna and transfected with 100 ng of plasmids expressing indicated sirna resistant YFP fusions of full length or RING deleted (_BCPS) TRIM15 or YFP alone Data represent the mean ± SD of three experiments carried out in triplicates. Statistical significance values * corresponds to p< Movie S1. Time-lapse differential interference contrast imaging of wound healing assay. Confluent monolayer of HT1080 cells were treated with control non-targeting sirna (NTsi) (top row) 2
7 or TRIM15 sirna without or with ectopic expression of sirna resistant TRIM15 (middle and bottom rows respectively). The cells were imaged from 0 to 13 h after wounding the monolayer. Tracked individual cells (colored dots) and their migration trajectories (colored lines) are shown in the right panels. Note the smooth trajectories for control and TRIM15si rescue panels compared to random migratory pattern of TRIM15 sirna treated cells. In addition, TRIM15 sirna treated cells display multiple lateral filopodia compared to a dominant leading edge exhibited by control and TRIM15sitreated rescued cells. See also Fig. S3 B-F. Scale bar: 100 μm. Movie S2. TRIM15-depletion impairs two dimensional migration in HeLa cells. HeLa cells were treated for 48 h with control NT sirna (NTsi; top panel) or TRIM15 sirna (T15si) panel) without or with ectopic expression of sirna resistant TRIM15-YFP for rescue (middle and bottom panels respectively). The cells were replated on fibronectin-coated coverslips and rested for h before time-lapse differential interference contrast imaging with a 10 X objective lens. The first frame in the rescue panel shows an image acquired in the YFP channel (red) to identify the cell expressing sirna resistant TRIM15-YFP. At this magnification, FAs cannot be seen and TRIM15-YFP expressing cells are identified by the presence of fluorescent cytoplasmic bodies. Tracked individual cells (colored dots) and their migration trajectories (colored lines) are shown as overlays. Note the restricted migration trajectories of TRIM15-depleted cells compared to long tracks displayed by control and the rescued cell. The longer migration trajectories seen, if any, in TRIM-depleted cells is a consequence of cell rounding, detachment and reattachment due to cell division. Scale bar: 10 μm. Movie S3. A comparison of FA dynamics in control and TRIM15-depleted cells under steadystate conditions. HeLa cells stably expressing zyxin-yfp and treated for h with control NT (NTsi; left panel) or TRIM15 (T15si; right panel) sirna were subjected to time-lapse microscopy for 2-3 h to monitor FA dynamics. Examples of FA assembly and disassembly events are highlighted using green and red boxes respectively. FA assembly and disassembly rates were calculated from 6-8 time lapse movies encompassing >200 FAs using Focal Adhesion Analysis Server. See also Fig. 6 C, D. Scale bar: 10 μm. 3
8 Movie S4. A comparison of microtubule-induced FA disassembly in control and TRIM15- depleted cells. HeLa cells stably expressing zyxin-yfp and treated with control NT (NTsi; left panel) or TRIM15 (T15si; right panel) sirna were treated with 10 μm nocodazole for 2-4 h to inhibit microtubule polymerization and prevent FA disassembly. The cells were then imaged for 2 h immediately after nocodazole was washed out to monitor FA disassembly. Note the rapid disassembly of FAs in control cells compared to that seen for TRIM15 sirna treated cells. An example of FA disassembly event is shown in the boxed region. FA disassembly rates were calculated from atleast 6 movies for each condition encompassing ~175 FAs. See also Fig. 6L. Scale bar: 10 μm. Movie S5. Disassembly modes of TRIM15 and Zyxin at focal adhesions. HeLa cells stably expressing TRIM15-YFP (left panel) or Zyxin-YFP (right panel) were subjected to time-lapse microscopy for 90 min at steady-state to monitor the way they disassemble from FAs. The red arrow heads point to disassembling FAs. Note that TRIM15 disassembles from FAs by collapsing into an oligomeric structure, to some extent instantaneously whereas zyxin dissipates slowly into the actin network and cytoplasm during disassembly. Scale bar: 10 μm Supplementary References: Jones, D. T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. Journal of molecular biology 292,
9 Movie 1. Movie 2.
10 Movie 3. Movie 4. Movie 5.
11 Paxillin amino acid Peptide Sequence co-ordinates FIHQQPQSSSPVYGSSAK VGEEEHVYSFPNK ELDELMASLSDFK KPIAGQVVTAMGK Table S1. Details of peptides derived from paxillin that were identified by mass spectrometric analyses of TRIM15 immunoprecipitates. Constructs Source Reference CFP fusions of human zyxin and paxillin Pekka Lappalainen (University of Helsinki, (Hotulainen and Lappalainen, 2006) Finland) CFP fusion of Human Talin1 Pietro De Camilli (Yale University, New Haven, CT) (Lee et al., 2005). Lentivirus expression construct for mtagrfpttubulin CT) Table S2. Details of constructs used in the study. Brett Lindenbach (Yale University, New Haven, (Counihan et al., 2011) sirna/shrna Sequence Source TRIM15 sirna #1 CCCUGAAGGUGGUCCAUGAUU Dharmacon Inc. TRIM15 sirna #2 GCGAGAACGAUGCCGAGUUUU Dharmacon Inc. Paxillin sirna CCCUGACGAAAGAGAAGCCUAUU Dharmacon Inc control non-targeting CUUACGCUGAGUACUUCGAAATT Samchully Pharm CO., (NT) luciferase sirna LTD (Seoul, Korea) TRIM15 shrna#1 CAGCGGTTGTTTTTACTTT OpenBiosytems Inc TRIM15 shrna#2 AGCGGTTGTTTTTACTTTA OpenBiosytems Inc control non-targeting TACAAACGCTCTCATCGACAAG OpenBiosytems Inc. (NT) luciferase shrna Table S3. List of sirna/shrna used in the study and their sources
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SUPPLEMENTARY INFORMATION FILE Existence of a microrna pathway in anucleate platelets Patricia Landry, Isabelle Plante, Dominique L. Ouellet, Marjorie P. Perron, Guy Rousseau & Patrick Provost 1. SUPPLEMENTARY
Mannen et al., http :// /cgi /content /full /jcb /DC1
Supplemental material JCB Mannen et al., http ://www.jcb.org /cgi /content /full /jcb.201601024 /DC1 THE JOURNAL OF CELL BIOLOGY Figure S1. Characterization of SNB components. (A) SNB localization of Venus-tagged
Expanded View Figures
EMO reports rystal structure of Mis18 Yippee-like domain Lakxmi Subramanian et al Expanded View Figures Figure EV1. Structural characterization of the N-terminal Yippee-like globular domain of spmis18.
This is the author's accepted version of the manuscript.
This is the author's accepted version of the manuscript. The definitive version is published in Nature Communications Online Edition: 2015/4/16 (Japan time), doi:10.1038/ncomms7780. The final version published
Supplementary Figure 1 Pfn1, but not other Pfn isoforms are expressed in
Supplementary Figure 1 Pfn1, but not other Pfn isoforms are expressed in platelets. (a) RT-PCR of Pfn isoforms in control mouse platelets, Pfn1 -/- platelets and control heart. Expected band size for Pfn1
J. Cell Sci. 128: doi: /jcs : Supplementary Material. Supplemental Figures. Journal of Cell Science Supplementary Material
Supplemental Figures Figure S1. Trio controls endothelial barrier function. (A) TagRFP-shTrio constructs were expressed in ECs. Western blot shows efficient Trio knockdown in TagRFP-expressing ECs. (B)
Supplementary Figure 1 Phosphorylated tau accumulates in Nrf2 (-/-) mice. Hippocampal tissues obtained from Nrf2 (-/-) (10 months old, 4 male; 2
Supplementary Figure 1 Phosphorylated tau accumulates in Nrf2 (-/-) mice. Hippocampal tissues obtained from Nrf2 (-/-) (10 months old, 4 male; 2 female) or wild-type (5 months old, 1 male; 11 months old,
Xu et al., Supplementary Figures 1-7
Xu et al., Supplementary Figures 1-7 Supplementary Figure 1. PIPKI is required for ciliogenesis. (a) PIPKI localizes at the basal body of primary cilium. RPE-1 cells treated with two sirnas targeting to
Supplementary Figure 1 PARP1 is involved in regulating the stability of mrnas from pro-inflammatory cytokine/chemokine mediators.
Supplementary Figure 1 PARP1 is involved in regulating the stability of mrnas from pro-inflammatory cytokine/chemokine mediators. (a) A graphic depiction of the approach to determining the stability of
Supplementary Figure S1 Purification of deubiquitinases HEK293 cells were transfected with the indicated DUB-expressing plasmids.
Supplementary Figure S1 Purification of deubiquitinases HEK293 cells were transfected with the indicated DUB-expressing plasmids. The cells were harvested 72 h after transfection. FLAG-tagged deubiquitinases
Supplementary methods
Supplementary methods Cell culture, infection, transfection, and RNA interference HEK293 cells and its derivatives were grown in DMEM supplemented with 10% FBS. Various constructs were introduced into
Nature Structural & Molecular Biology: doi: /nsmb.3018
Supplementary Figure 1 Validation of genetic complementation assay in Bmal1 / Per2 Luc fibroblasts. (a) Only Bmal1, not Bmal2, rescues circadian rhythms from cells. Cells expressing various Bmal constructs
monoclonal antibody. (a) The specificity of the anti-rhbdd1 monoclonal antibody was examined in
Supplementary information Supplementary figures Supplementary Figure 1 Determination of the s pecificity of in-house anti-rhbdd1 mouse monoclonal antibody. (a) The specificity of the anti-rhbdd1 monoclonal
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(Supplementary Methods and Materials) GST pull-down assay GST-fusion proteins Fe65 365-533, and Fe65 538-700 were expressed in BL21 bacterial cells and purified with glutathione-agarose beads (Sigma).
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SUPPLEMENTAL DATA SUPPLEMENTAL FIGURE LEGENDS SUPPLEMENTAL FIGURE S1. Identification of BmCREC. (A) Amino acid sequences of BmCREC show the peptides identified in LC-MS/MS analysis (marked by red letters
Figure S1. Verification of ihog Mutation by Protein Immunoblotting Figure S2. Verification of ihog and boi
Figure S1. Verification of ihog Mutation by Protein Immunoblotting Extracts from S2R+ cells, embryos, and adults were analyzed by immunoprecipitation and immunoblotting with anti-ihog antibody. The Ihog
Supplementary Figures
Supplementary Figures 11 1 1 Supplementary Figure 1. Clinical features of EBS cases with KLHL mutations. (a) Photo of Patient 1 s hands showing mild atrophic skin and unaffected finger nails. (b) The skin
VCP adaptor interactions are exceptionally dynamic and subject to differential modulation by a VCP inhibitor
VCP adaptor interactions are exceptionally dynamic and subject to differential modulation by a VCP inhibitor Liang Xue 1, Emily E. Blythe 1, Elyse C. Freiberger 2, Jennifer Mamrosh 1, Alexander S. Hebert
Nature Medicine doi: /nm.3554
SUPPLEMENTARY FIGURES LEGENDS Supplementary Figure 1: Generation, purification and characterization of recombinant mouse IL-35 (ril-35). High-Five insect cells expressing high levels of the bicistronic
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doi:10.1038/nature10016 Supplementary discussion on binding site density for protein complexes on the surface: The density of biotin sites on the chip is ~10 3 biotin-peg per µm 2. The biotin sites are
T H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Kunduri et al., http://www.jcb.org/cgi/content/full/jcb.201405020/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. Subcellular localization and interactions of PAPLA1.
MEFs were treated with the indicated concentrations of LLOMe for three hours, washed
Supplementary Materials and Methods Cell Fractionation MEFs were treated with the indicated concentrations of LLOMe for three hours, washed with ice-cold PBS, collected by centrifugation, and then homogenized
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doi:10.1038/nature11070 Supplementary Figure 1 Purification of FLAG-tagged proteins. a, Purification of FLAG-RNF12 by FLAG-affinity from nuclear extracts of wild-type (WT) and two FLAG- RNF12 transgenic
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SUPPLEMENTARY INFORMATION Supplementary figures Supplementary Figure 1: Suv39h1, but not Suv39h2, promotes HP1α sumoylation in vivo. In vivo HP1α sumoylation assay. Top: experimental scheme. Middle: we
Supplementary Fig. 1 Proteomic analysis of ATR-interacting proteins. ATR, ARID1A and
Supplementary Figure Legend: Supplementary Fig. 1 Proteomic analysis of ATR-interacting proteins. ATR, ARID1A and ATRIP protein peptides identified from our mass spectrum analysis were shown. Supplementary
42 fl organelles = 34.5 fl (1) 3.5X X 0.93 = 78,000 (2)
SUPPLEMENTAL DATA Supplementary Experimental Procedures Fluorescence Microscopy - A Zeiss Axiovert 200M microscope equipped with a Zeiss 100x Plan- Apochromat (1.40 NA) DIC objective and Hamamatsu Orca
seminal vesicle thymus kidney lung liver
a 1 HEK293 1 B 3 3 T GFPSMAD TGFβ (T)/ BMP (B) IB: SMAD1/3TP IB: GFP b wild type mouse tissue kidney thymus seminal vesicle liver lung brain adipose tissue muscle pancreas heart uterus spleen testis IB:
DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration
DDA3 associates with microtubule plus ends and orchestrates microtubule dynamics and directional cell migration Liangyu Zhang 1,2,4, Hengyi Shao 1,4, Tongge Zhu, Peng Xia 1, Zhikai Wang 1,2, Lifang Liu
To examine the silencing effects of Celsr3 shrna, we co transfected 293T cells with expression
Supplemental figures Supplemental Figure. 1. Silencing expression of Celsr3 by shrna. To examine the silencing effects of Celsr3 shrna, we co transfected 293T cells with expression plasmids for the shrna