Phospho acetyl coa carboxylase p acc

After the treatment with metformin (2 mM), glucose (15 mM) and/or siAMPK (50 nM)/siControl (50 nM) or Compound C (1 μM) at 24 h or 48 h, the cells were lysed in ice-cold NP-40 buffer containing protease inhibitor (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride). The whole-cell lysates were incubated with protein A/G Sepharose beads (Roche) for 2 h at 4 °C, and the beads were discarded to eliminate non-specific binding. Then, the supernatants were incubated overnight with a Bcl-xl antibody (Cell Signaling Technology, Danvers, USA) at 4 °C, followed by incubation with protein A/G Sepharose beads for another 2 h at 4 °C. Then, western blotting was performed with the indicated antibodies according to standard protocols as previously described^{20 (link)}. The primary antibodies, including p-AMPK (T172) (#50081), phospho-Acetyl-CoA carboxylase (p-ACC) (#11818), phospho-Tuberin/TSC2 (p-TSC2) (Ser1387) (#23402), phospho-Raptor (p-Raptor) (Ser792) (#2083), p-mTOR (S2448) (#5536), phospho-p70 S6 kinase (p-S6K1) (Thr389) (#97596), p-ULK1 (Ser757) (#14202), p-4E-BP1 (Thr70) (#9455), cleaved Caspase-3 (Cl-Caspase3) (Asp175) (#9664), p-Akt (Ser473) (#4060), p-Akt (Thr308) (#13038), RagB (#8150), LC3B (#3868), β-Actin (#3700) were all purchased from Cell Signaling Technology, Danvers, USA.

Sample preparation, protein separation, transfer and immunolabelling was performed as in [20 (link)]. Briefly, cells were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 10% SDS, 1% Nonidet P-40, 1 mM Na₃VO₃, and 1 mM NaF, 0, 5% sodium deoxycolate, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture, pH 8.0). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% acrylamide gels and blotted onto nitrocellulose membranes. After blocking in 5% (w/v) non-fat dry milk, the membranes were washed with 1X TWEEN-TBS and incubated with primary antibodies for overnight at 4°C. For the detection of AMPK activity phospho-acetylCoA carboxylase (pACC) (1:500), (Cell Signaling) was used. Actin was detected using a rabbit polyclonal antibody (Sigma, 1:5000). The secondary antibody was IgG peroxidase HRP conjugate (Sigma, 1:2000). Bands were visualized by enhanced chemiluminescence reaction (West Pico ECL Kit, Thermo Scientific).