Fig. 7: CASK-Mint1 interaction regulates Munc18-1 membrane localization.

a Loss of CASK (CASK Ri-1) or Mint1 (Mint1 Ri-1) reduced the membrane localization of Munc18-1 which was detected by immunofluorescence using anti-Munc18-1 (green) (upper panel). Represented peak curve diagrams showing cellular distribution on membrane (m) and in cytoplasm of Munc18-1 immunofluorescent signals measured by Image J with linear scanning (lower panel). b Subcellular fractionation confirmed the increase of Munc18-1 in cytosolic fraction. and decrease in membrane fraction (M) when CASK or Mint1 was silenced (Ci & Mi). Con, control; T, total lysate. Ecadherin, LaminB and Tubulin were used as markers for membrane, nucleus and cytosol respectively (middle panel). The signals of Munc18-1 were quantified using a densitometer (right panel). c Overexpressing Mint1 4A mutant but not wild type (WT) Mint1 disrupted the membrane localization of Munc18-1 which was detected by immunofluorescence using anti-Munc18-1 (green) (upper panel). Represented peak curve diagrams showing cellular distribution on membrane (m) and in cytoplasma of Munc18-1 immunofluorescent signals measured by Image J. with linear scanning (low panel). *For a and c cells were treated with glucose. *For a and c, Z-projection in the X–Z direction and in the Y–Z direction are shown. The green and red lines indicate the orthogonal planes of the X–Z and Y–Z projection, respectively. *Note the different distribution pattern of Munc18a in different situations showed by the low panels of a and c. d Fractionation assays by gel filtration chromatography revealed co-fractionation of CASK, Mint1 and Munc18 in INS-1E cells transfected with non-silencing control siRNA (NC), CASK-specific siRNA (CASK Ri-1) or Mint1-specific siRNA (Mint1 Ri-1). After being stimulated with glucose, the lysate was fractionated by gel filtration chromatography using a Superose 6 10/300 GL column. The western blot images were showed for the representative fractions of Paek1 and Peak2 from INS-1E cell transfected with NC (i), Mint1 Ri-1(ii) and CASK Ri-1(iii). Eluted protein profiles from each fraction were quantified and normalized to T (total protein) for CASK, Mint1 and Munc18 (iv-vii) in INS-1E cells transfected with NC, CASK Ri-1 or Mint1 Ri-1, respectively. e The hypothetical model.