Ab151318

After treatment with rapamycin, hGFs were harvested and lysed with RIPA Lysis and Extraction buffer (Pierce Biotechnology, Rockford, IL, USA) and Halt Protease and Phosphatase Inhibitor (Pierce Biotechnology). Protein concentration was quantified using a BCA protein assay kit (Pierce) and fractionated by sodium dodecyl sulphate-polyacryalamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Co., Bedford, MA, USA). The membrane was blocked with 5% nonfat milk and 2% BSA in TBST for 1 h at room temperature and incubated with certain primary antibodies, rabbit anti-human p21 antibody (ab109520; Abcam, Cambridge, UK), rabbit anti-human p16 antibody (ab108349; Abcam), rabbit anti-human phospho-p70 S6 kinase antibody (Thr421/Ser424) (Cell Signaling Technology), rabbit anti-human p70 S6 kinase antibody (Cell Signaling Technology), and rabbit anti-human β-tubulin antibody (ab151318; Abcam) at 4°C overnight. The membrane was washed 4 times and incubated with a 1 : 3000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG antibody (R&D Systems) for 1 h at room temperature. After being washed 3 times with TBST, the immunodetection was developed with the ECL-chemiluminescent kit (Thermo Scientific).