Rabbit anti pck1

For Western blotting, frozen tissue was pulverized in liquid nitrogen and homogenized in 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS containing Complete EDTA-free Protease Inhibitor Cocktail (Sigma) and PhosSTOP phosphatase inhibitor cocktail (Sigma). Tissue homogenates were spun down @ 4 °C (16,000 g for 10 min) to remove debris. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher). Homogenates were diluted with SDS sample buffer and boiled for 5 min. Equal amounts of protein were loaded and separated by SDS-PAGE using a Criterion electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA), transferred onto the polyvinylidene fluoride membrane, and immunoblotted with specific antibodies: rabbit anti-albumin (Bio-Rad; #AHP1478); rabbit anti-Pck1 (Abcam; ab28455); or rabbit anti-GAPDH (#2118; Cell Signaling Technologies, Danvers, MA, USA).