Proteome profiler adipokine array kits

Adipokine protein expression levels in serum (n=3 per genotype) or eWAT (n=6–7 per genotype) were assessed using mouse Proteome Profiler Adipokine Array kits (R&D Systems; Minneapolis, MN; catalog# ARY013) according to manufacturer’s instructions. Briefly, eWAT was homogenized in PBS with 10 μg/mL each aprotinin, leupeptin, and pepstatin. After homogenization, Triton X-100 was added to each sample at 1% final concentration. Samples were frozen at −80°C, thawed, and centrifuged at 10,000 × g for 5 minutes at 4°C to remove cellular debris. Sample protein concentrations were quantified using a bicinchoninic acid assay with bovine serum albumin as the standard (Sigma-Aldrich, St. Louis, MO). Serum assays were performed using 100ul of mouse serum as specified in the kit instructions. Membranes were incubated with chemiluminescence reagents and then exposed to autoradiography film for times ranging from one to five minutes. Film from the five-minute exposure was used for densitometry analyses, which were performed with Image Studio Lite software (Version 3.1; Li-Cor; Lincoln, NE). Densitometry values for each adipokine were normalized to values for the reference spot on each membrane. Resulting values were then compared between genotypes.