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2 protocols using rabbit anti phospho paxillin

1

Immunofluorescence and Immunoprecipitation Protocols

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Cells were fixed in methanol/acetone (1:1) with exception of samples processed for Rac-GTP labeling, which were fixed in 2% formalin and permeabilized with 0.1% Triton X-100. Processing of samples was carried out as described previously.19 (link) Antibodies used for immunofluorescence were: rabbit anti-GIT219 (link); rabbit anti-DOCK5 (described above); rabbit anti-DOCK1 (Santa Cruz Biotechnology, H-70); mouse anti-paxillin (BD Biosciences, cl. 349), mouse anti-ARP3 (Sigma, cl. FMS338); mouse anti-Rac-GTP (NewEast Biosciences, 26903); and mouse anti-Myc epitope (Cell Signaling Technologies, clone 9B11). Confocal images were acquired using a spinning disk confocal microscope.
Antibodies used for immunoprecipitation and western blotting were: mouse anti-Crk (BD Biosciences; clone 22); rabbit anti-Crk (Santa Cruz Biotechnology; C-20), mouse anti-DOCK1 (Santa Cruz Biotechnology; clone H-4), mouse anti-p130Cas (BD Biosciences; clone 21), mouse anti-Rac1 (BD Biosciences; clone 102), rabbit anti-Rac3 (Abcam; clone EPR6680), rabbit anti-FAK (Santa Cruz Biotechnology; clone A-17), rabbit anti-phospho-p130Cas(Tyr410), rabbit anti-phospho-paxillin(Tyr118) and rabbit anti-phospho-FAK(Tyr397). All phospho-specific antibodies were from Cell Signaling Technologies.
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2

Immunofluorescence and Immunoprecipitation Assays

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Cells were fixed in methanol/acetone (1:1) with exception of samples processed for Rac-GTP labeling, which were fixed in 2% formalin and permeabilized with 0.1% Triton X-100. Processing of samples was carried out as described previously.19 (link) Antibodies used for immunofluorescence were: rabbit anti-GIT2;19 (link) rabbit anti-DOCK5 (described above); rabbit anti-DOCK1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, clone H-70); mouse anti-paxillin (BD Biosciences, San Jose, CA, USA, clone 349), mouse anti-ARP3 (Sigma, St Louis, MO, USA, clone FMS338); mouse anti-Rac-GTP (NewEast Biosciences, Malvern, PA, USA, clone 26903); and mouse anti-Myc epitope (Cell Signaling Technologies, Danvers, MA, USA, clone 9B11). Confocal images were acquired using a spinning disk confocal microscope.
Antibodies used for immunoprecipitation and western blotting were: mouse anti-Crk (BD Biosciences; clone 22); rabbit anti-Crk (Santa Cruz Biotechnology; C-20), mouse anti-DOCK1 (Santa Cruz Biotechnology; clone H-4), mouse anti-p130Cas (BD Biosciences; clone 21), mouse anti-Rac1 (BD Biosciences; clone 102), rabbit anti-Rac3 (Abcam; clone EPR6680), rabbit anti-FAK (Santa Cruz Biotechnology; clone A-17), rabbit anti-phospho-p130Cas(Tyr410), rabbit anti-phospho-paxillin(Tyr118) and rabbit anti-phospho-FAK(Tyr397). All phospho-specific antibodies were from Cell Signaling Technologies.
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