Trans blot turbo midi polyvinylidene fluoride

Approximately 50 mg of mouse liver was homogenized in radio immunoprecipitation assay lysis buffer (MilliporeSigma, Burlington, MA) containing the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The homogenates were centrifuged at 10,000g for 10 minutes at 4°C to obtain liver lysates, and the protein concentrations were measured with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The liver lysates (20 μg of protein) were subjected to 4%‐15% Criterion TGX Precast Midi Protein Gel (Bio‐Rad, Hercules, CA) and transferred to Trans‐Blot Turbo Midi polyvinylidene fluoride (Bio‐Rad) using the Trans‐Blot Turbo Transfer System (Bio‐Rad). Membranes were blocked with 5% OmniPur bovine serum albumin, fraction V (MilliporeSigma) in a mixture of Tris‐buffered saline and 0.1% Tween 20 (MilliporeSigma) and incubated overnight with primary antibodies against c‐Myc (sc‐41, 1:500 dilution; Santa Cruz Biotechnology, Dallas, TX) and CDK4 (11026‐1‐AP, 1:1,000 dilution; Proteintech Group, Rosemont, IL). The β‐actin band was obtained by reprobing the membranes with antibody against β‐actin (#8457, 1:1,000 dilution; Cell Signaling Technology, Danvers, MA) and was used as a loading control. Each band intensity was quantified using Bio‐Rad Image Lab software, normalized by those of the loading control, and expressed as a fold change relative to WT mice.