Integrin α5

Equal amounts of proteins were separated by SDS-PAGE, and then transferred onto polyvinylidene membranes (Millipore, SaintQuentin enYvelines, Belgium) by electrotransfer. Membranes were blocked with 5% skim milk in PBS-T (containing 0.1% Tween-20), and proteins of interest were visualized using specific Anti-Stathmin (Cell SignalingTechnology, Beverly, MA, USA), poly (ADP-ribose) polymerase (PARP) (BD Biosciences, San Jose, CA, U.S.A.), integrin β1, and integrin α5(BD Biosciences, San Jose, CA), caspase-3 (Santa CruzBiotech, CA, USA). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody and secondary antibodies, conjugated with horseradish peroxidase (HRP), were ordered from KangChen Biotech (Shanghai, China).
For immunoprecipitation, the cells were lysed inbuffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet™ P-40 (NP-40), 5 mM EDTA, 5 mM ethylene glycol tetraacetic acid, 15 mM MgCl2, 60 mM β-glycerolphosphate, 0.1 mM sodiumorthovanadate, 0.1 mM NaF, 0.1 mM benzamide, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF. Twenty microliters of protein A/Gagarose beads (BD Bioscience Pharmingen) were added to the lysates for proper periods of incubation. The beads were then washed and subjected to SDS-PAGE and immunoblotting.