Irdye 800 coupled goat anti rabbit secondary antibody

For western blot analysis of adiponectin, serum samples (n = 2 male mice) were diluted in PBS plus sample loading buffer followed by boiling for 10 min at 95°C. For western blot analysis of tissue samples (n = 2–3 male mice), proteins were extracted in RIPA buffer. Samples were loaded on a Criterion precast gel (Bio-Rad, Hercules, CA), and after SDS-PAGE the samples were subjected to immunoblot analysis with PVDF membrane (Millipore, Bedford, MA) using polyclonal anti-adiponectin^{60 (link)}, C/EBPα, pAkt (Ser473), tAkt, pErk1/2 (Thr202/Tyr204) and tErk1/2 (#8178, #9271, #9272, #9101 and #9102 Cell Signaling, Danvers, MA) and β-actin (Sigma, St. Louis, Mo) antibodies (1:300 dilution for adiponectin, 1:1000 dilution for all other antibodies). For Western blots in Fig. 3b and 3d, Secondary antibodies used were an IRDye 800-coupled goat anti-rabbit secondary antibody (Rockland, Gilbertsville, PA) and an IRDye 700-coupled goat anti mouse secondary antibody (Rockland, Gilbertsville, PA) (for labelling of mouse IgG as serum loading control). The membrane was scanned by the LI-COR Odyssey infrared imaging system at 700- and 800-nm channels simultaneously. For other western blots, ECL method was used and the PVDF membranes were exposed to X-film. All the unprocessed original scans can be found in the Supplementary Fig. 9.