Journal: Genes & Development
Article Title: cAMP promotes pancreatic ?-cell survival via CREB-mediated induction of IRS2
Figure Lengend Snippet: CREB promotes insulin/IGF signaling via induction of IRS2. ( A ) Western blot analysis of IRS1 and IRS2 levels in MIN-6 cells treated with forskolin for increasing times in serum-supplemented medium. Cells were infected with A-CREB or control GFP adenovirus as indicated. ( B ) Transient transfection assay of 293T cells with IRS2 promoter construct or control somatostatin CRE luciferase plasmid. Cells treated with forskolin or DMSO vehicle. Cotransfection with A-CREB expression plasmid is indicated. ( C ) Chromatin immunoprecipitation assay of the IRS2 promoter using CREB 244 antiserum. Presence of the IRS2 promoter or negative control GAPDH in CREB immunoprecipitates determined by PCR amplification of anti-CREB or control IgG immunoprecipitates. ( D ) IRS2 expression is disrupted in A-CREB transgenic mice. ( Top ) Immunohistochemical staining of pancreatic sections from wild-type and RIP A-CREB transgenic mice using anti-IRS2 antiserum. ( Bottom ) Western blot assay of islet-cell extracts from wild-type and A-CREB mice using anti-IRS2 antiserum. Comparable levels of CREB protein in transgenic and wild-type lysates. ( E ) cAMP-dependent induction of IRS-2 potentiates growth factor signaling to Akt. Effect of IGF1 on Akt phosphorylation at Thr 308 in cells pretreated with forskolin or vehicle under serum-starved conditions for 8 h. Effect of A-CREB on IGF-stimulated Akt Thr 308 phosphorylation.
Article Snippet: PCR was performed with primers complementary to the IRS2 promoter region (forward –1244/–1225, reverse –1092/–1106) or to the GAPDH gene (forward 586/605, reverse 1037/1018) as control, by using the GC-rich PCR Kit (Roche).
Techniques: Western Blot, Infection, Transient Transfection Assay, Construct, Luciferase, Plasmid Preparation, Cotransfection, Expressing, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Amplification, Transgenic Assay, Mouse Assay, Immunohistochemistry, Staining