Mouse monoclonal antibody against rac1

RAC1 activation was analyzed using a GTPase activation assay kit purchased from Upstate Biotechnology according to the manufacturer's instructions. Briefly, cell lysates were incubated with 10 μg of glutathione S-transferase- (GST-) agarose beads coupled to the GST-Pak-1-binding domain (PBD). Samples were subjected to SDS-PAGE and immunoblotting. Membranes were probed with a mouse monoclonal antibody against RAC1 (Abcam) followed by western blot analysis. The amount of PBD-bound RAC1 was normalized to the amount of protein in cell lysates.

After 48 hours of transfection, cells were subjected to protein extraction using cell lysis buffer containing 1% protease inhibitors. Protein concentrations of the homogenized lysates were measured using a BCA protein assay kit (Sangon, Shanghai, China). Aliquots containing 30 μg of protein were separated by 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal antibody against integrin β6 (1:1000; Millipore, CA, USA), mouse monoclonal antibody against Rac1 (1:300; Abcam, Hong Kong), or anti-β-actin (1:1000; ZSGB-Bio, Beijing, China), followed by secondary antibodies (1:2000; goat-anti rabbit or mouse IgG, ZSGB-Bio) conjugated to peroxidase for 1 hour at room temperature. After washing, immunoreactivity was visualized using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.