Fig. 2. Active CDC42 alleles diminish the association of IQGAP1 with the exocyst. (A) Effects of different alleles of CDC42 on the co-immunoprecipitation of IQGAP1 with EXO70. Lysates from pancreatic β-cells transiently expressing equal amounts of different HA-tagged CDC42 alleles (bottom) were used to co-immunoprecipitate IQGAP1 (middle panel) with EXO70 (top panel) monoclonal antibodies. IQGAP1 was blotted from 5% of the input protein (lower panel) as a loading control. Cells expressing the vector alone were used as a positive control and mock is a negative control with anti-Myc antibodies. (B) Quantitation of IQGAP1 association with EXO70 in the presence of CDC42 mutants. The intensity of the IQGAP1 bands co-precipitated with EXO70 in the presence of the CDC42 alleles were quantified by densitometry using Quantity One software and a ChemiDoc XRS system (Bio-Rad), and are expressed as the mean ± s.d. for n=3. (C) Binding of CDC42 to IQGAP1 is necessary for the negative effects of CDC42 on the co-immunoprecipitation of IQGAP1 and EXO70. Co-immunoprecipitation of IQGAP1 and EXO70 was performed from cells expressing either a HA-CDC42-F28LC37A double mutant that diminishes CDC42 binding to IQGAP1 (see D, top), or expressing the mutant V5-IQGAP1-FMK, which lacks the 24 amino acids of the CDC42-binding region (see D, bottom), and from their parental F28L or V5-F1 proteins. The histogram represents the mean ± s.d. for n=3 blots. (D) Confirmation of the binding abilities of the mutants used in C. The double-mutant HA-CDC42-F28LC37A, the deletion-mutant V5-IQGAP1-FMK and their parental constructs transiently expressed in βTC-6 cells were used for IP with antibodies for the respective tags as shown on the figure and probed for co-immunoprecipitation of IQGAP1 (top) or CDC42 (bottom). The input is presented in the lower part of each panel. WB, western blot; WCL, whole cell lysate.