Molecular Regulation of Insulin Secretion

Cytokine treatment of cells was performed with 5 ng/ml IL1β from R&D systems. RNA isolation and reverse transcription followed by quantitative real-time PCR (qRT-PCR) were performed (Qiagen miScript system) [6 (link)]. miRNA or mRNA expression was quantified relative to U6 or β-actin, respectively, using the comparative Ct method (Primer sequences in Supplementary Table 1) [6 (link)]. Pulldown of mRNAs bound to 50 nM biotinylated miR-21-3p, miR-21-5p, or control Caenorhabditis elegans miR-67 was performed as described [27 ]. Luciferase assays were performed using a Gaussia luciferase/secreted alkaline phosphatase dual reporter system (GeneCopoeia) and wild-type rat Tgfb2 3′ untranslated region (UTR) and Smad2 3′UTR or mutated 3′UTRs for Tgfb2 (positions 1281–1289) and Smad2 (positions 8900–8908) [6 (link)]. Immunoblotting was performed as described, visualized using an Odyssey imaging system, and quantified by LI-COR software (LI-COR Biotech) (antibodies in Supplementary Table 2) [6 (link)].
Static GSIS and perifusion were performed as described with supernatants assayed for insulin using ELISA (Cisbio) and normalized to total DNA content (PICO Green Assay; Invitrogen) [28 (link), 29 (link)].