Western Blotting Analysis of Signaling Proteins

CProteins were extracted in RIPA buffer supplemented with phosphatase and proteinase inhibitors. Protein concentration was determined with the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Details on Western blotting procedures were previously described [34 (link)]. In brief, equal quantities of protein were resolved using SDS-PAGE and transferred onto nitrocellulose membranes, blocked in 5% dry fat milk, incubated with primary antibodies at 4°C and subsequently with corresponding secondary antibodies at room temperature. Developed membranes were imaged with a ChemiDoc MP Imaging System (BioRad, Hercules, CA, USA) and quantified with ImageLab software (BioRad, version 5.2.1). The following primary antibodies were used (diluted 1:1’000 except Actin, which was diluted 1:5’000): pERK1/2, #9101; p-p38, #9212, pSTAT3, #9145; ATGL, #2138; pHSL #4139 (all from Cell Signaling); Gapdh, G9545 (Sigma-Aldrich), Actin, MAB1501 (Millipore), GLUT1 and GLUT4 (gift from Dr. A. Klip, The Hospital for Sick Children, Toronto, ON, Canada; samples were not boiled for Western blots assessing GLUT1 and GLUT4 protein levels).