Fig. 5: Kindlin-2 interacts with and stabilizes MafA to activate insulin gene expression.

a IF staining. Pancreatic sections of 2-month-old male control (RIP-Cre) and K2-RIP mice were stained with the indicated antibodies. Scale bar, 50 μm. b qPCR analysis. Total RNA isolated from 2-month-old control and K2-RIP islets was subjected to qPCR analysis. Insulin mRNA was normalized to Gapdh mRNA. *P < 0.05, versus control. c IHC. Sections of 2-month-old pancreas were stained with the indicated antibodies. Scale bar, 50 μm. N = 5 per genotype. d qPCR analysis. Total RNA from b was used. e (top) Schematic representation of the rat Ins1 gene promoter. e (bottom) COS-7 cells were transfected with p460rIns1-luc or p460rIns1C1mt-luc, pRL-SV40, and expression plasmids for β-gal, Kindlin-2, MafA, or Kindlin-2 plus MafA. After 48 h, cells were harvested for dual-luciferase assays. *P < 0.05, versus β-gal, ^#P < 0.05, versus MafA. f–k IP assay. Whole cell extracts from COS-7 cells overexpressing pCMV/Flag-Kindlin-2 and pCMV/HA-MafA (f, g) or INS-1 cells (h, i) or a mixture of GST-MafA and GST-Kindlin-2 (j, k) were immunoprecipitated with the indicated antibodies, followed by Western blot analyses using the indicated antibodies. l A schematic showing the ___domain structure of Kindlin-2 and the deletion mutants. m COS-7 cells were co-transfected with p460rIns1-luc, pRL-SV40 (for normalization), pCMV/MafA, and pCMV vectors expressing wild type (WT) or various deletion Kindlin-2 mutants, followed by dual-luciferase assays. *P < 0.05, versus pCMV/β-gal. n IP assay. COS-7 cells were transfected with MafA expression plasmids and various Flag-Kindlin-2 deletion mutant expression plasmids. After 48 h, whole cell extracts were prepared and immunoprecipitated with M2 antibody, followed by Western blot analysis using M2 (top) or MafA antibody (bottom) (left panel). o COS-7 cells were transfected with a MafA expression plasmid with or without Kindlin-2 overexpression and treated with 10 µg/ml cycloheximide, followed by Western blotting at the indicated times. p Western blot analysis. Cytoplasmic (CE) and nuclear extracts (NE) from INS-1 cells subjected to Western blotting for Kindlin-2, MafA, and tubulin (a cytoplasmic protein). Results are expressed as mean ± standard deviation and Student’s t test was used in this figure. Source data for b, d–k, m–p are provided as a Source Data file.