Sds polyacrylamide gels

Reduced and heat-denatured samples were separated on 10% SDS-polyacrylamide gels (acrylamide from National Diagnostics). For native gel electrophoresis, 5% polyacrylamide gels were prepared without SDS. Additionally, SDS and reducing agents were eliminated from the running and sample loading buffers. Following gel electrophoresis, proteins were transferred to nitrocellulose membranes in transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. Traditional immunoblotting procedures were followed, and results were visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (Thermo Scientific, Rockford, IL). Primary antibodies included: the anti-adiponectin antibody from Thermo Scientific (Rockford, IL); phospho-AMPKα (Thr172), phospho-Akt (Ser473), and MCP-1 antibodies purchased from Cell Signaling Technology, Inc. (Danvers, MA); and anti-MAPK (Erk 1/2) antibody from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). Optical densities of all protein bands were analyzed using Image Studio Lite software (Licor Biosciences, Lincoln, NE).