Hrp conjugated anti β actin

The frozen AT specimens were homogenised in a urea/SDS buffer supplemented with Complete Protease Inhibitor (Roche Applied Science). Samples (80 μg total protein) were run on a 7% acrylamide gel and western blotting (WB) was conducted as previously described [7 (link)]. Ten samples were run per gel with groups mixed. A rabbit anti-mouse HIF-1α Primary antibody (10006421 Cayman) and HRP-conjugated anti-β-actin (Abcam) was used for detection. Densitometry was performed using ImageJ, and the sample HIF1A abundance was corrected for actin. Due to technical difficulties of achieving high quality and quantifiable western blot images, samples had to be re-run on several occasion which led to sample depletion. For this reason the final number of successful quantifications was less than total participants. Gene expression in adipose tissue samples was determined as described [8 (link)].

Protein samples (75 μg) were analyzed by 12.5% SDS-PAGE. The proteins were transferred onto nitrocellulose membranes (0.2 μm, Bio-Rad, USA) using blotting buffer (7.25 mg/ml Tris base, 3.6 mg/ml glycine, and 0.5 mg/ml SDS) and a blotting machine (Trans-Blot SD semi-dry transfer cell, Bio-Rad, USA) at 18 V for 30 min. The protein-containing membranes were washed twice for 5 min with PBS-Tween solution. The blotted membranes were blocked using 5% skim milk in a PBS-Tween solution at 4°C overnight. The specific proteins were detected by adding a primary antibody solution (rabbit anti-CAECAM at 1:500 dilution (Abcam, UK) and mouse anti-β-actin at 1:3,000 dilution (Sigma, USA)) to the blotted membranes, incubating for 10 hr and washing three times for 5 min each with a PBS-Tween solution. The secondary antibody solution (HRP-conjugated anti-β-actin (Abcam, UK) and anti-rabbit IgG for anti-CAECAM (Sigma, USA) at 1:1,000 dilution) was added to the blots, incubated for 2 hr and washed three times for 5 min each with a PBS-Tween solution. Enhanced chemiluminescence was employed using chemiluminescent reagents (Thermo Scientific, USA). The blots were detected and analyzed by Image Quant LAS 4000 software (GE Healthcare Life Science, UK).