Anti nov ccn3

Frozen adipose and liver tissues were homogenized and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease and phosphatase inhibitors (Complete TM Mini and PhosSTOP TM, Roche Diagnostics, Indianapolis, IN, USA). Protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide (SDS) gels and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After blocking, the membranes were then incubated at 4 °C overnight with the following primary antibodies: anti-UCP-1, anti-PRDM16, anti-FGF21, anti-Sirt1, anti-PGC1α, anti-pAKT, anti-AKT, anti-pACC, anti-ACC, anti-pIRS1 Ser³⁰⁷, anti-NOV/CCN3, anti-TWIST2, anti-HIF1α, anti-β-actin (Cell Signaling Technology, Danvers, MA, USA), anti-pIR tyr⁹⁷² (Millipore, Bedford, MA, USA), anti-HO-1 (Enzo Life Sciences, Farmingdale, NY, USA). Membrane incubations were carried out using a secondary infrared fluorescent dye conjugated antibody absorbing at both 800 nm and 700 nm. The blots were visualized using an Odyssey Infrared Imaging Scanner (Li-Cor Science, Lincoln, NE, USA)) and quantified by densitometric analysis after normalization with β-actin. Results were expressed as optical density (O.D.) as previously described [19 (link)].