Western Blot Analysis of Adipose and Liver Proteins

Western blots of proteins in the adipose and liver were performed as previously described (Berg & Scherer, 2005 (link); Collins et al., 2016 (link); Hotamisligil, 2006 (link)). Primary antibodies for anti-NOV/CCN3, anti-IL-6, anti-pP65, anti-P65, anti-MMP9, anti-MMP2, anti-MT1-MMP, anti-TIMP1, anti-TIMP2, anti-PRDM16, anti-PGC-1α, anti-SOD1, anti-TWIST1, anti-SIRT1, anti-Mfn1, anti-FGF21, anti-CREG1, anti-UCP1, anti-pAKT, anti-AKT, and anti-β-actin were purchased from Cell Signaling Technology, Danvers, MA, USA. The anti-pIR tyr⁹⁷² antibody was purchased from Millipore, Bedford, MA, USA. The anti-HO-1 antibody was purchased from Enzo Life Sciences, Farmingdale, NY, USA. The secondary antibodies labeled with either IRDye 680 or IRDye 800 were purchased from LICOR Biosciences, Lincoln, NE. Immunoreactivity was visualized and quantified by infrared scanning in the Odyssey system (LICOR Biosciences, Lincoln, NE) and quantified after normalization with β-actin and expressed as arbitrary units (AU).