P p70s6k1

HepG2 cells were harvested and boiled with a sample buffer (1 × DB). Proteins were separated by SDS-PAGE and transferred to Poly Vinylidene Di-Fluoride (PVDF) membranes. After blocking with 3% bovine serum albumin or 3% skim milk for 1 h, the membranes were incubated with a primary antibody overnight at 4 °C. After being washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20, the membranes were reacted with an antimouse or rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000) for 1 h at room temperature. Membranes were washed with PBS three times and subjected to immunoblotting using SuperSignal West Pico Substrate (Thermo Scientific, Waltham, MA, USA) or ImmunoStar LD (Wako). Band intensities were quantitatively analyzed using ImageJ. The following antibodies were used: actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), AMPK (#5832, Cell Signaling Technology, Danvers, MA, USA), p-AMPK (#2535, Cell Signaling Technology), ACC (#3662, Cell Signaling Technology), p-ACC (#11818, Cell Signaling Technology), p53 (sc-393031, Santa Cruz Biotechnology), p-p53 (#9284, Cell Signaling Technology), p70S6K1 (#2708, Cell Signaling Technology), p-p70S6K1 (#9234, Cell Signaling Technology), cleaved caspase-3 (#9664, Cell Signaling Technology), caspase-3 (#9665, Cell Signaling Technology).

WST-1 assay kit was purchased from Daeillab (Daeillab, Korea). and dihydrotestosterone (DHT) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Specific antibodies such as Bcl-2, Bax, APAF-1 was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as t-mTOR, p-mTOR, LC3, p62 Cyclin E1, p-cdk2, t-p70 S6K, p-p70 S6K1, t-4E-BP1, p-4E-BP1 were obtained from Cell Signaling Technology (Beverly, USA). and Specific antibodies such as β-actin and Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse Cell Cycle Kit (MCH100106) and Muse Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany).