Fak y397

An amount of 5 μg of each antibody was used to precipitate integrin subunits from 250 μg of total resistance artery radioimmunoprecipitation assay extract. The antibodies used for immunoprecipitation were: β1 (AB1952), β3 (AB1932) and α5 (aba5b) integrin antibodies (Chemi-Con, Moses Lake, Wash., USA). Agarose IgG/protein A (Sigma-Aldrich, UK) was used to bind and identify coprecipitated complexes. Subsequent Western blot analysis was performed using FAK Y397 (1:1,000) antibody and Src Y418 (1:500, 0.5 μg/ml; Abcam). Protein A-HRP was used for chemiluminescent detection of coprecipitated proteins [22 (link)].

The 4G10 antibody for phosphotyrosine was purchased from Millipore (Boston, MA). The antibodies for paxillin, p130Cas, and p130Cas Y249 were purchased from BD Biosciences (Franklin Lakes, NJ). The antibodies for paxillin Y31, FAK Y397, and FAK Y576/577 were purchased from Abcam (Cambridge, UK). The antibodies for paxillin Y118 was purchased from Cell Signing Technology (Boston, MA) and FAK from Proteintech (Rosemont, IL). Alexa Flour 546 phalloidin and Alexa Fluor 488 goat anti-mouse IgG were purchased from Life Technologies (New York, USA). HRP Conjugated goat anti-rabbit IgG, and HRP Conjugated goat anti-mouse IgG were purchased from CW Biotech (Beijing, China).
Human umbilical vein endothelial cell line ECV-304 was maintained in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO₂.