Anti akt antibody

Cultured cells or tissues were harvested and lysed with RIPA lysis buffer (Beyotime, China) for 30 min on ice. After centrifugation at 12,000 g for 20 min, the concentration of proteins was measured using Bradford's reagent (Bio-Rad laboratories, USA). The protein samples were denatured by boiling for 10 min and loaded onto SDS–PAGE gel for electrophoresis. The proteins were transferred onto PVDF membrane (Millipore, USA) which was then incubated in the blocking solution (5% FBS) at room temperature for 1 h. The anti-REPS2 antibody (Abcam, UK) or anti-RalBP1 antibody (Santa Cruz, USA), anti-RAC1 antibody (GeneTex, USA), anti-CDC42 antibody (GeneTex, USA), anti-MMP9 antibody (Proteintech, USA), anti-MMP2 antibody (Proteintech, USA), anti-cyclinD1 antibody (Abcam, UK), anti-EGFR antibody (Abcam, UK), anti-pEGFR antibody (Abcam, UK), anti-AKT antibody (Abcam, UK), anti-pAKT antibody (Abcam, UK), anti-Erk antibody (Abcam, UK) and anti-pErk antibody (Abcam, UK) was added into blocking solution and incubated at 4°C overnight. The membranes were subsequently incubated with HRP-labeled goat anti-rabbit IgG for 1.5 h at room temperature. Protein expression was normalized against GAPDH expression (RD, USA) or β-actin expression (Bioworld, USA). Bands were visualized by employing the BeyoECL Plus DetectionSystem (Beyotime, China) and Bio-Rad Image Lab Software (CA, USA).