Anti akt

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 total protein was prepared using cell lysates (Beyotime, Shanghai, China), and the concentration of protein was determined with the BCA protein assay kit (Thermo, Massachusetts, USA). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose filter membrane. Membranes were treated with 5 % skimmed milk powder (Biolab, Beijing, China) and incubated with primary antibodies at 4 °C overnight, and then secondary antibodies (Immunoway, Suzhou, China) at room temperature for 1.5 h. The following antibodies were purchased: anti-FGF6 (Immunoway, SuZhou, China), anti-GAPDH (Immunoway, SuZhou, China), anti-AKT (Immunoway, SuZhou, China), anti-P-AKT (Immunoway, SuZhou, China), anti-ERK1/2 (Immunoway, SuZhou, China), anti-P-ERK1/2 (Immunoway, SuZhou, China), anti-FGFR (Immunoway, SuZhou, China), and anti-P-FGFR (Immunoway, SuZhou, China). After an appropriate amount of 1:10,000 diluted Secondary antibody incubated, ECL reagent (FuDeBio, Hangzhou, China) was used to detect the bands, and Quantity One (Bio-Rad, Hercules, CA) was used to analyze the positive bands.