Fig 5.

The complex consisting of IQGAP1 and GDP-bound Rab27a regulates endocytosis. (A) MIN6 cells expressing phogrin-Cherry together with GFP or GFP-Cdc42-T17N were incubated with 20 mM glucose for 5 min. (B) The percentage of transfected cells that had internalized phogrin-Cherry was analyzed by determination of the percentage of transfected cells that displayed cytoplasmic distribution of phogrin-Cherry fluorescence. (C) COS-7 extracts expressing Flag-Rab27a-T23N, either the G12V-Cdc42 or the T17N-Cdc42 mutant, and GFP-IQGAP1-WT or GFP-IQGAP1-GRD were immunoprecipitated with an anti-GFP antibody. The immunocomplexes were analyzed by immunoblotting using anti-GFP, anti-Flag, and anti-T7 antibodies. The percentage of input protein coimmunoprecipitated was 2.0%. (D) COS-7 extracts expressing Flag-IQGAP1, GFP-IQGAP1-GRD, and GFP-Rab27a-T23N were immunoprecipitated with an anti-Flag antibody. The immunocomplexes were analyzed by immunoblotting using anti-Flag and anti-GFP antibodies. The percentage of input protein coimmunoprecipitated was 0.2%. −, GFP. (E) MIN6 cells expressing phogrin-Cherry and either GFP or GFP-IQGAP1-GRD were incubated with 20 mM glucose for 5 min and were analyzed by immunofluorescence. Bars, 10 μm. (F) The percentage of transfected cells that had internalized phogrin-Cherry was analyzed by determination of the percentage of transfected cells that displayed cytoplasmic distribution of phogrin-Cherry fluorescence. The data in panels B and F were statistically analyzed as described in the legend to Fig. 3C. (G) Schematic model of IQGAP1 function in membrane trafficking.