Polyclonal ARHGAP25 antibody was prepared from rabbit serum after intracutaneous
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1 Preparation and purification of polyclonal antibodies Polyclonal ARHGAP25 antibody was prepared from rabbit serum after intracutaneous injections of glutathione S-transferase-ARHGAP25-( ) (GST-coiled coil) fusion protein. For affinity purification GST and GST-coiled coil protein were prepared from bacteria and bound to glutathione-agarose beads (Sigma). (Bead preparation: suspend beads in 50 volumes of NETN (0.5% NP-40, 20 mm Tris, ph 8.0, 100 mm NaCl, 1 mm EDTA), spin down beads (1 min at 2000 rpm), add 10 volumes of NETN, incubate overnight at 4 C on rocker, spin down beads, take off supernatant and add 1 bead volume of NETN). GST-beads and GST-coiled coil beads were washed three times with 10 ml NETN buffer. Then beads were washed twice with 0.1 M borate buffer ph 8.0, once with 0.1 M borate buffer ph 9.0 and then once with 0.2 M borate buffer ph 9.0. After washing, beads were incubated in 40 mm dimethypimelinidate (dissolved in 0.2 M borate buffer ph 9.0) in a rotary shaker for 1 hour at 4 C. Beads were washed twice with 0.1 M borate buffer ph 8.0, and then incubated with 40 mm ethanolamine (in 0.1 M borate buffer ph 8.0) in a rotary shaker for 45 min at 4 C. Then beads were washed three times with cold PBS, once with 0.2 M glycine-hcl, ph 2.5, once with 1 M K 2 HPO 4, then twice with PBS. After washing, GST-beads were incubated with 4 ml of serum diluted with 4 ml of PBS containing 0.2% (w/v) Tween20 in a rotary shaker overnight at 4 C. Beads were washed three times with PBS containing 0.2% (w/v) Tween20. Thereafter, supernatant was added to GST-coiled coil coated beads and after rotation for 2 hours, were loaded onto a column and washed three times with 10 ml of PBS containing 0.2% (w/v) Tween20, then washed twice with 10 ml of PBS. Antibody was eluted with 0.2 M glycine-hcl, ph 2.5 into a microcentrifuge tube containing 1 M K 2 HPO total fractions were collected. Protein concentration was determined as described by Bradford 1 and
2 peak fractions were pooled. Antibody was dialyzed overnight against 1000 ml of PBS/glycerol (PBS:glycerol is 1:1) at 4 C. The specificity of anti-arhgap25 antibody was validated with western blot analysis of recombinant, GST-fusion fragments of ARHGAP25 (Figure S1), the overexpression of ARHGAP25R192A and ARHGAP25 in COSphoxFcγR cells (Figure 3A) and PLB-985 cells treated with ARHGAP25-specific shrna (Figure 4B). Anti-p50RhoGAP polyclonal antibody was prepared as described. 2 REFERENCES 1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: Sirokmany G, Szidonya L, Kaldi K, Gaborik Z, Ligeti E, Geiszt M. Sec14 homology domain targets p50rhogap to endosomes and provides a link between Rab and Rho GTPases. J Biol Chem. 2006;281(9): Figure S1. Testing of polyclonal anti-arhgap25 antibody (A) GST-fusion full-length ARHGAP25 or its indicated fragments were separated with SDS/PAGE and immunoblotted with ARHGAP25-specific anti-coiled coil antibody. In the first lane we found a strong signal at 83 kda, which is the GST-tagged full-length ARHGAP25, and some other, weaker signals at lower molecular weight, which are the proteolytic fragments of the full-length protein. The antibody also recognised specifically the GST-coiled coil domain. The PH domain and the GST protein also reacted weakly with the
3 antibody, which is caused by the presence of polyclonal anti-gst antibody contamination. (B) Specific anti-gst antibody was used as a control that recognised all samples. Figure S2. Verification of the critical arginine in ARHGAP25 Effect of full-length wild-type or mutated ARHGAP25 on in vitro [γ- 32 P]GTP-hydrolysis of Rac. Mean ± S.E.M. of three separate experiments is shown. *, p<0.05 vs. control. Figure S3. ARHGAP25 colocalizes with endogenous Rac in COS7 cells Cyan fluorescent protein-coding control vector (A-C), CFP-tagged ARHGAP25 (D-F) or CFP-ARHGAP25R192A (G-I) overexpressed by COS7 cells are shown in blue. Endogenous Rac was labelled with monoclonal anti-rac1 antibody (BD Biosciences) and detected with Alexa-488 conjugated anti-mouse-ig secondary antibody (green). C,F,I, merge. Figure S4. Enrichment of wild-type and GAP-deficient ARHGAP25 around the phagocytosed yeast particles. CFP-ARHGAP25R192A (B,E) and CFP-ARHGAP25 (C,F) shows enrichment around the phagosomes of COSphoxFcγR cells, whereas the cyan fluorescent protein localizes in the cytosol and mainly in the nucleus (A,D). The same cells are shown both in colour (A-C) and in black-and-white (D-F). In A-C p40 Phox is shown in green, wild-type or mutant CFP- ARHGAP25 (or only CFP) in blue and yeast particles in red. Enrichment of ARHGAP25 around the phagosomes is indicated by arrows. Figure S5. Neutrophilic differentiation of PLB-985 cells causes significant increase in CD11b expression
4 Non-treated (A), control shrna-treated (B) and ARHGAP25-silenced (C) PLB cells were stained with R-phycoerythrin-labelled monoclonal anti-cd11b antibody (Dako), and CD11b expression was measured by flow cytometry. Black line, non-labelled cells; blue line, nondifferentiated PLB cells labelled with anti-cd11b; red line, differentiated PLB cells labelled with anti-cd11b. Representative experiments are shown.
5 construct GST-ARHGAP25 full length GST-PH domain GST-GAP domain GST-PH+GAP domain GST-Coiled coil domain ARHGAP25R192A CFP-ARHGAP25 primer pairs CGGGATCCGTGATGACTGGCGAGCAGATGGCTG CCGCTCGAGCGAGCCTCGGTCTTGGGTCC CGGGATCCCCCATGTCCCTCGGTCAGTCGGCC GGAATTCGCCAAACAC TCCACAGGGTGTGC CGCGGATCCGCTGGCACACCCTGTGGAGTG GGAATTCTGACAGGGGTATATCCTTGGAC CGGGATCCGTGATGACTGGCGAGCAGATGGCTG GGAATTCTGACAGGGGTATATCCTTGGAC CGGGATCCAACTCTGAAACTGGGCCTGG CCGCTCGAGCCTTAAGCCTCGGTCTTGGGTTC GAAGAGGGCATCTTCGCTCTTCCTGGGCAGGAC GTCCTGCCCAGGAAGAGCGAAGATGCCCTCTTC AACTCGAGACATGTCCCTCGGTCAGTC TTGGATCCTAAGCCTCGGTCTTGGGTTC ARHGAP25 shrna/sirna 5'-3' ACCGGGAAGTTTGTCTTTGAAATTCAAGAGATTTCAAAGACAAACTTCCCTTTTTC control shrna/sirna 5'-3' ACCGGGAAGTTTGTCTTGTAAATTCAAGAGATTTACAAGACAAACTTCCCTTTTTC p50rhogap-cfp CCGCTCGAGGCCATGGATCCGCTCTCAGAGCTGCAGG GGAATTCGGAGCCCGCTGGGGTCCGGGCTTG
6 Figure S1 A B M (kda) M (kda) anti-coiled coil anti-gst
7 Figure S2 GST-Rac ]GTP (%) Protein bo ound [ 32 P 120 n = 3 GST-Rac ±S.E.M. + f.l. ARHGAP ARHGAP25R192A Time (min)
8 CFP Rac merge FigureS3 A B C D E F G H I ARHGAP25R192A ARHGAP25 control vector
9 Figure S4 control vector ARHGAP25R192A ARHGAP25 merged A B C CFP (m monochro ome) D E F
10 A non-treated PLB-985 Figure S5 non-labelled PLB non-diff. PLB+αCD11b diff. PLB+ α CD11b CD11b B control shrna treated PLB-985 non-labelled PLB non-diff. PLB+αCD11b diff. PLB+ α CD11b CD11b C ARHGAP25 shrna treated PLB-985 non-labelled PLB non-diff. PLB+αCD11b diff. PLB+ α CD11b CD11b
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