Anti human β actin

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).