Enhanced chemiluminescence detection system

Tissues were mechanically disrupted using a Polytron homogenizer (Glen Mills Inc., USA) and lysed on ice in 1 % Nonidet P-40 in buffer (50 mM Hepes, pH 7.2, 150 nM NaCl, 0.5 % sodium deoxycholate, 0.1 % SDS, 1× complete protease inhibitors (Roche, Switzerland) for 30 min followed by sonication. Insoluble fragments were removed by centrifugation (20,000g, 20 min). Protein concentrations were measured by BCA (Pierce, Rockford, IL). Clear lysates (20–50 µg) were separated on 4–20 % polyacrylamide gels and electro-transferred to PVDF membranes (Millipore, USA) in Towbin buffer (25 mM Tris, 192 mM glycine) supplemented with 10 % methanol. The membranes were blocked in 10 % skimmed milk overnight and incubated with primary antibody [Ab285 or anti-actin (C-11, Santa Cruz Biotechnolgy, USA)] for 60 min. After washing the membranes were incubated for 30 min with secondary horseradish-conjugated, anti-rabbit IgG or anti-goat antibody (Santa Cruz Biotechnology, USA) and developed by enhanced chemiluminescence detection system (Roche, Switzerland).

For Western blot analysis, total protein extraction from scWAT biopsies was performed using an ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. First, the total protein content was measured using the Bradford protein assay. Thus, 30 µg of extracted proteins were loaded on 10% SDS–PAGE and transferred to PVDF membranes (3,010,040,001, Amersham, USA) by electroblotting using the BioRad transfer system. The membranes were blocked with 5% nonfat dry milk in PBST (phosphate-buffered saline containing 0.01% Tween-20) for 1 h at room temperature. Next, they were incubated in a 1:500 and 1:100 dilution of the primary mouse anti-human CD31 (Santa Cruz, USA) 4 and primary rabbit antibody anti-human β-actin (ab227387, Abcam Inc., Cambridge, MA) antibodies, respectively, overnight at 4 ºC. Next, the membranes were washed with PBST three times and incubated with a 1:2000 dilution of horseradish peroxidase-conjugated secondary goat anti-mouse IgG (170–6516, Bio-Rad). Finally, the Western blot bands were pictured following an enhanced chemiluminescence detection system (11,500,708,007, Roche, USA) on radiography films. Then, the photos were scanned and analyzed using Image J software (v.1.41).