Anti p70s6

After treatment, HepG2 cells were lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing 1 mmol/l phenylmethylsulfonyl fluoride. As previously described [26 (link)], the protein concentration in each sample was measured with a Bio-Rad detergent-compatible protein assay (Bio-Rad laboratory, Tokyo, Japan). Samples containing 10 μg of protein were resolved by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad), followed by incubation with primary antibodies targeting cyclin D, cyclin-dependent protein 4(CDK4), and β-actin [28 (link)]. After incubation with the secondary antibody (horseradish peroxidase-conjugated sheep anti-rabbit IgG, 1:20,000), reaction products were detected with an Amersham ECL Plus system and an enzyme-linked chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ, USA), and the band density was quantified using a LumiVision Analyzer (Aisin, Kariya, Japan). The following primary antibodies were used: anti-cyclin D, anti-p21, anti-p53, anti-p70S6, anti-cleaved caspase3, anti-caspase3, and anti-β-actin (all from Cell Signaling Technology, Beverly, MA, USA), anti-Cdk4, anti-sodium glucose co-transporter 1, and anti-sodium glucose co-transporter 2 (al from Abcam, Cambridge Science Park, Cambridge, UK).

Anti-LC3B, P62, type I collagen and BNIP3 antibodies were purchased from Abcam (Cambridge, MA); anti-α-tubulin antibody from Sigma (St. Louis, MO), and anti-p70S6, p-mTOR and mTOR antibodies from Cell Signaling Technology (Danvers, MA). MitoSOX Red, Mitotracker Deep Red, Alexa 488 and Alexa 633-conjugated secondary antibodies were obtained from Invitrogen (Carlsbad, CA).

Media for cell culture, anti-hemagglutinin (HA) polyclonal antibody, and LipofectAMINE Plus reagent were ordered from Invitrogen (Carlsbad, CA, USA). Anti-AMPKα (AMPK α-subunit), anti-phosphorylated AMPKα (Thr 172), anti-ACCα, anti-phospho-ACCα, anti-p70S6, and anti-phospho-p70S6 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Monoclonal anti-HA was from Roche Molecular Biochemicals (Indianapolis, IN, USA). The anti-myc antibody and the polyclonal anti-HA antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-human synphilin-1 polyclonal antibody was generated using the human synphilin-1 fragment (34–500 aa) as the antigen as previously described [3 (link)]. Anti-actin antibody was from Sigma (St. Louis, MO, USA). Compound C (an AMPK inhibitor) [40 (link)] was from Sigma (Burlington, MA, USA).