Fig 3.
IQGAP1 is required for the glucose-induced redistribution of Rab27a. (A) IQGAP1-silenced MIN6 cells were analyzed by immunoblotting with anti-IQGAP1, anti-coronin 3 (Coro3), antiactin, and anti-Rab27a antibodies. (B) IQGAP1-silenced MIN6 cells expressing Flag-Rab27a were incubated with 3 or 20 mM glucose (G3 or G20, respectively) for 5 min. The cells were immunostained with anti-Flag (green) and anti-IQGAP1 (red) antibodies. siControl, control siRNA. (C) The percentage of cells analyzed as in panel B with a peripheral distribution of Flag-Rab27a was determined. The results of statistical analysis of the percentage of transfected cells that had Rab27a near the plasma membrane are shown. More than 40 randomly selected cells (more than 8 cells/experiment) were examined. Data are expressed as means ± SD from 4 independent experiments. The asterisk indicates a significant difference (P, <0.01) from siControl cells stimulated with 3 mM glucose by the unpaired Student t test. (D) IQGAP1-silenced MIN6 cells expressing GFP-IQGAP1 or GFP-RNAiR-IQGAP1 were analyzed by immunoblotting using anti-IQGAP1 and antiactin antibodies. −, GFP. (E) IQGAP1-silenced MIN6 cells expressing Flag-Rab27a and GFP or GFP-RNAiR-IQGAP1 were incubated with 20 mM glucose for 5 min. The cells were immunostained with an anti-Flag antibody (red). Control, GFP. Bars, 10 μm. (F) The percentage of cells analyzed as in panel E with a peripheral distribution pattern of Flag-Rab27a was determined. Statistical analysis was performed as described for panel C. Control, GFP. The asterisk indicates a significant difference (P, <0.01) from siControl-expressing cells by the unpaired Student t test.