Fig. 5.

PI4KIIα regulates insulin secretion via a PKD-dependent pathway. A Proteins biotinylated by BirA*-PI4KIIα or BirA*-PI4KIIIβ in MCF-7 cells were purified using streptavidin agarose. The immunoprecipitates were immunoblotted with antibodies against PKD, Myc, and GAPDH. B MIN6 cells were transfected with or without RFP-PI4KIIα, and the nucleus (blue), PKD (green), and insulin (red or magenta as indicated) were immunostained using DAPI or the respective antibodies. Scale bar, 2 μm. C MCF-7 cells were transfected with GFP-PKD. After 24 h, the cells were lysed in RIPA buffer and subjected to pull-down with exogenous GST, GST-PI4KIIα WT, or GST-PI4KIIαK152A expressed in E. coli. D MIN6 cells were transfected with GFP, GFP-PI4KIIα, or GFP-PI4KIIαK152A. After 30 h, insulin secretion in response to 3.3 or 33 mmol/L glucose stimulation with or without 0.2 μmol/L TPA was measured using an insulin ELISA kit. E MIN6 cells were transfected with control siRNA or mouse PI4KIIα siRNA for 60 h; then, insulin secretion in response to 3.3 or 33 mmol/L glucose stimulation with or without 10 μmol/L CID755673 was measured using an insulin ELISA kit. The data are presented as the mean ± SD of three independent experiments, and all the experiments were performed three times in triplicate. *p < 0.05