Western Blot Analysis of Adiponectin Protein

Previously described procedures were used [51 (link)]. Briefly, protein samples (20 µg) were separated using 10% SDS-PAGE gels, then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% nonfat dry milk in TBS containing 0.1% Tween for 1 h at room temperature and then blotted with primary antibodies (anti-Adiponectin (1:500) (#2789) (Cell Signaling Technology, Inc., Beverley, MA, USA), anti-β-tubulin (1:1000) (sc-9104) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight. After washing, membranes were incubated with a secondary horseradish peroxidase (HRP)-coupled antibody and visualized using Immobilon HRP substrate (Millipore). The density of the bands was quantified using ImageJ Software (National Institute of Health, Bethesda, MD, USA). The ratio of the intensity of the target protein to that of β-tubulin loading control was calculated to represent the expression level of the protein.