Supersignal west pico plus chemiluminescence system

Hepatic and adipose tissue proteins were extracted using RIPA lysis and extraction buffer (Thermo Scientific, Rockford, IL) containing protease inhibitor cocktail (Millipore Sigma, Burlington, MA). For all tissues, 20 μg of total homogenate protein was analyzed. For plasma protein analysis, 2 μl of plasma was employed. The proteins were subjected to SDS PAGE in 12% gels followed by a transfer onto PVDF membrane. Immunoblotting was performed using rabbit polyclonal antibodies directed against mouse cytochrome P450 isoform 2B10 (CYP2B10, 1:5000, EMD Millipore, Temecula, CA), cytochrome P450 isoform 26A1 (CYP26A1, 1:1000, Bioss, Woburn, MA), retinol-binding protein 4 (RBP4, 1:3000), and peroxisome proliferator-activated receptor gamma (PPARγ, 1:1000, Santa Cruz Biotechnology, CA). Protein loading and relative quantification of protein expression were normalized using β-actin as a reference protein (1:10,000; Millipore Sigma, St. Louis, MO). Protein bands were visualized using Super Signal West Pico PLUS chemiluminescence system (Thermo Scientific, Rockford, IL), films were digitally scanned and analyzed using ImageJ (National Institutes of Health) to generate quantitative relative protein expression data [79 (link)].