G6pase α

Whole liver was immediately frozen using clamps pre-cooled to the temperature of liquid nitrogen and stored at −80° for Western blot analysis. Briefly, samples were analyzed for protein content, and separated on 4%–12% Bis-Tris gels at 150 volts for 1.5 h. Transfer was for 2 h at 30 volts on ice. Blots were incubated with primary antibody to GR (1:1000; Cell Signaling Technology, Danvers, MA, USA), PEPCK (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 11β-HSD1 (1:1000; Abcam Cambridge, MA, USA), G6Pase α (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and G6Pase β (1:200; Santa Cruz Biotechnology Inc.) overnight at 4°C. To probe for actin, blots were incubated with anti-actin primary antibody (1:5000; EMD Millipore, Billerica, MA, USA) for 1 h at room temperature. After washing, blots were incubated with secondary antibody, anti-rabbit Immunoglobulin G (IgG) (heavy and light chains) DyLight (1:100,000; Cell Signaling Technology) and simultaneously with anti-mouse IgG (H + L) DyLight (1:15,000; Cell Signaling Technology), for 1 h at room temperature. Images of membranes were obtained with the abundance of all proteins of interest normalized to actin, which was the internal control. Band density was analyzed using Odyssey-Clx (LI-COR, Lincoln, NE, USA) and Image Studio (LI-COR).