Dimethyloxalylglycine dmog

Western blotting was performed using antibodies against ACC (Cell Signaling Technologies (CST), ACC2 isoform (CST), actin (Sigma), AMPKα (CST), HA (CST), Flag (Sigma), hydroxyproline (Abcam), phospho-ACC (CST), phospho-AMPKα (CST), PHD3 (Novus Biologicals) and tubulin (Sigma). 1% NP40 buffer containing protease and phosphatase inhibitors was used to prepare lysates, unless otherwise indicated. For ACC hydroxylation time course studies, the pan-PHD inhibitor dimethyloxalylglycine (DMOG, Frontier Scientific, 1 mM) was added to the lysis buffer to prevent further hydroxylation in the lysate. For immunoprecipitation of endogenous ACC2, cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 % NP40, 0.1 mM DTT, 0.05 mM DMOG, protease inhibitors (Roche) and 100 ul of phosphatase inhibitor 2/3 (Sigma). Cell lysates were centrifuged for 15 minutes to remove debris. Then, 2 ug of ACC antibody (CST) or ACC2 antibody (CST), was added to the cleared cell lysate (500 ng of protein) and immunoprecipitated with EZview Red Protein G Affinity resin (Sigma). Transiently overexpressed HA- or Flag-tagged proteins, lysates were immunoprecipitated using EZview anti-HA Affinity resin (Sigma) or anti-Flag Affinity resin (Sigma).