Pepck c h 300

Liver lysates were made by homogenizing pulverized, freeze-clamped liver tissue in lysis buffer (50 mM Tris-HCl pH 7.4, 1 mM EGTA, 150 mM NaCl, 5 mM MgCl_2, 1% NP-40) with added cOmplete protease inhibitor tablet (Roche) per 50 mL buffer. SDS samples were prepared by adding 2x SDS sample buffer (20% glycerol, 120 mM Tris-HCl pH 6.8, 4% SDS, 0.02% bromophenol blue, 4% β-mercaptoethanol) to one volume of lysate and boiling at 95°C for 5 min. Samples were run on Novex 4–12% Tris-Glycine gels (Invitrogen) and gels were transferred to PVDF membranes by semi-dry transfer. Antibodies used to determine protein expression were: PEPCK-C H-300 (Santa Cruz Biotechnology, Cat. No.: sc-32879), PCB H-2 (Santa Cruz Biotechnology, Cat. No.: sc-271493), Phospho-CREB Ser133 87G3 (Cell Signaling, Cat. No.: 9198), CREB 48H2 (Cell Signaling, Cat. No.: 9197), Phospho-AMPKα Thr172 40H9 (Cell Signaling, Cat. No.: 2535), AMPKα (Cell Signaling, Cat. No.: 2532), GAPDH D16H11 XP^® (Cell Signaling, Cat. No.: 5174), G6Pase (Santa Cruz Biotechnology, Cat. No.: sc-27198), Phospho-ACC (Cell Signaling, Cat. No.: 3661), and ACC (Cell Signaling, Cat. No.: 3676)