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Fig. 7. IQGAP1 influences protein synthesis and exocytosis. (A) Differential effects of IQGAP1 domains on insulin exocytosis. Early passages of βTC-6 cells (Beta) stably expressing similar levels of the V5-IQGAP1 constructs (on the x-axis) were selected to measure insulin exocytosis (ng/ml) in duplicate. Means ± s.d. for n=6 are shown. Values were normalized to cells that incorporated the vector (Beta-V), which were used as a control. For each construct, the left column represents the basal and the right column the glucose-induced insulin exocytosis. Statistical significance: P<0.001. (B) IQGAP1 influences protein synthesis. Early passages of βTC-6 clones stably expressing the indicated IQGAP1 domains were pulsed and chased/stimulated with glucose, as described in the Materials and methods. Upper panels: insulin antibodies were used to immunoprecipitate insulin from the labeled (left) and the chased (right) sets, resolved on a 20% SDS-PAGE and evaluated with a phosphorimager. Lower panel: western blot (WB) of 5% of the lysate from the 1-hour chased cells. The autoradiograph and the blot represent three experiments with identical results. (C) Immunofluorescence of insulin in cells stably expressing the IQGAP1 domains. Fixed βTC-6 cells stably expressing equal amounts of the indicated IQGAP1 domains were stimulated for 1 hour, fixed and stained with insulin guinea-pig polyclonal antibodies and Alexa-Fluor-488 goat anti-guinea-pig secondary antibodies, and the images were acquired at a 500-msec exposure time without post-image-acquisition processing.
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