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).

The human neuroblastoma cell line SK-N-MC was obtained from Korean Cell Line Bank (Seoul, Korea). The antibodies of p-Akt (Thr308), p-Akt (Ser473), Akt, mTOR, Caveolin-1, Flotillin-2, p-Tau (Ser396), Tau, p-NF-κB p65 (Ser536), NF-κB p65, Lamin A/C, CBP and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies of p-mTOR (Ser2448), p-p70S6K1 (Thr389) and p70S6K1, were acquired from Cell Signaling Technology (Beverly, MA, USA). Aβ, BACE1, HIF-1α and GPR40 antibodies were obtained from Abcam (Cambridge, MA, USA). The C99 antibody was purchased from EMD Millipore (Darmstadt, Germany). Horse radish peroxidase (HRP)-conjugated IgG was obtained from Jackson Immunoresearch (West Groove, PA, USA). PA, BSA, GW9508, ionomycin, PF4708671, LY294002 and rapamycin were purchased from Sigma Chemical Company (St. Louis, MO, USA). The Akt inhibitor, GW1100 and SN50 used here were purchased from Calbiochem (La Jolla, CA, USA). siRNAs for GPR40, GPR120, APP, BACE1, HIF-1α and non-targeting were obtained from Dharmacon (Lafayette, CO, USA).