Fig. 2: CASK formed a complex with mint1-munc18-1 upon glucose stimulation and regulated mint1-munc18 interaction.

a Immunomicrographs reveal membrane translocation of CASK in INS-1E cells following glucose stimulation (upper panel). Represented peak curve diagrams showing cellular distribution of CASK immunofluorescent signals measured by Image J with linear scanning (lower panel). b Subfractionation assay for membrane translocation of CASK upon glucose stimulation showed by western blot analysis (left panel). Quantification of the signal of CASKusing a densitometer (right panel). M: membrane fraction; C: cytosolic fraction; T: total lysate. c Overexpressing CASK/WT, CASK/S51/395A, CASK/S51/395D mutants in INS-1E cells. Immunomicrographs showing the subcellular localization of different CASK mutants. d Histograms showing expression of CASK-S51/395A mutant inhibited insulin secretion. e The effect of Mint1 on insulin secretion in INS-1E cells (left panel). Right panel, western blotting showing the efficiency of Mint1 knockdown (two distinct siRNA fragments, Ri-1 and Ri-2). f Western blot analysis showed that glucose-induced increased co-immunoprecipitation of endogenous CASK, Mint1 and Munc18-1 in a time-dependent manner in INS-1E cells. Non-immune serum (NS) was used as a negative control for immunoprecipitation. g The subcellular distribution of CASK, Munc18-1 and Mint1 before or after glucose stimulation. Cells were stained with anti-CASK (red) and anti-Munc18-1 (green) or anti-Mint1 (green) (left panel). Image pro plus was used to analyze the co-localization of the two proteins. Arrows indicates the membrane region. Note the overlap of the red and green signal peaks at the cell membrane after glucose stimulation (right panel).