The cell lysate for immunoblotting was extracted using radio-immuno precipitation assay (RIPA) lysis buffer (Cwbio, Beijing, China) with protease inhibitors (Roche, Germany) added. BCA Assay Kit (Cwbio) was used to quantify protein concentration. Protein sample mixed with a loading buffer (40 µg) was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for electrophoresis and transferred in a Tris-Glycine buffer to polyvinylidene fluoride (PVDF) membranes (Millipore, Danvers, USA). The PVDF membranes were blocked by 5% of defatted milk. The membranes were blocked and then incubated with specific primary antibodies for 12 h at 4 ℃. After washed thrice with Tris-buffered saline with Tween-20 (TBST), membranes were incubated with secondary antibody for 1 h at 37 ℃. Finally, immunoreaction signal was detected with enhanced chemiluminescence (ECL) solution (WBLUC0100, Millipore). Signal intensity of membranes was quantified using ImageJ software (Bio-Rad).
The antibody of anti-human PAK4 was purchased from Santa Cruz, California, USA, anti-Akt, anti-p-Akt, anti-mTOR, anti-LC3, anti-Tubulin, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-p53 from Immuno Way, Texas, USA, and horseradish peroxidase-conjugated secondary antibodies from Gene Tex, Texas, USA.
The antibody of anti-human PAK4 was purchased from Santa Cruz, California, USA, anti-Akt, anti-p-Akt, anti-mTOR, anti-LC3, anti-Tubulin, anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-p53 from Immuno Way, Texas, USA, and horseradish peroxidase-conjugated secondary antibodies from Gene Tex, Texas, USA.