Quantifying Insulin, RANKL, and OPG Levels

Insulin, hRANKL, and hOPG from serum or culture media were assayed using ELISA kits for mouse and human insulin (Mercodia Inc., Winston Salem, NC) (12 (link), 87 (link), 91 (link), 92 (link)), human RANKL (abcam, Waltham, MA), and human OPG (abcam), respectively. IL-1β and IL-6 production in monocytes was determined using ELISA, per the manufacturer’s instructions (BioLegend, San Diego, CA). For Western blot analysis, protein extracts (20 to 40 μg) from human and mouse islets treated with or without cytokines in the presence or absence of IgG, OPG (100 ng/ml), or DMB (100 ng/ml) for 24 hours were analyzed by immunoblotting using antibodies against p-NF-κB-Ser536, p-STAT1-Ser727 (Cell Signaling Technology, Danvers, MA), tubulin (EMD Millipore-Calbiochem, Burlington, MA), and glyceraldehyde-3-phosphate dehydrogenase (Sigma-Aldrich, St. Louis, MO) (table S4). Quantitative densitometry of digitalized blots was performed using the ImageJ program (12 (link), 87 (link), 88 (link), 90 (link)–92 (link)).