Western Blotting Technique for Protein Analysis

Western blots were performed as described^{42 (link)}. For protein preparation, cells or tissues were solubilized in a lysis buffer. Protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA). Then, 20 μg of the protein was electrophoresed by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). After blocking with 5% skimmed milk in Tris-buffered saline containing Tween 20 (0.1%) for 1 h, the membrane was incubated with polyclonal antibodies against phospho-ERK (1:1,000; Cell Signaling Tech., Danvers, MA, 4376S), ERK (1:1,000; Cell Signaling Tech., 4695S), Glut1 (1:1,000; AB chem, Cambridge, UK, ab652), adiponectin (1:1000; Cell Signaling Tech., 2789) or β-actin (1:1,000; Cell Signaling Tech., 4967S) at 4 °C with gentle shaking overnight. Antibodies were detected by horseradish peroxidase-linked secondary antibody (1:10,000; Cell Signaling Tech., 7074S), using the enhanced chemiluminescence Western Blotting Detection System (Molecular Imager Gel Doc XR System, BIO-RAD). The intensities of the protein bands were performed with ImageJ 1.52v software. Images of original western blotting are shown in Supplementary Figs. 1–3.