Fig. 3. The N-terminus of IQGAP1 interacts with EXO70 and SEPT2. (A) Schematic representation of the V5-His-tagged IQGAP1 constructs cloned into the pcDNA3.1 Topo vector and used for transient and stable expressions in this study: IQGAP1 full-length (F1), N-terminal (N1), C-terminal (C2) and the IR-WW domains. For abbreviations of the domains, see legend to Fig. 1. (B) Endogenous or exogenous IQGAP1 can co-immunoprecipitate two exocyst subunits and SEPT2 from different cell lines. Left: anti-IQGAP1 antibodies were used to co-immunoprecipitate SEC8, EXO70 and SEPT2 subunits from total lysate of the indicated cell lines and 5% of representative input lysate was blotted as a loading control; Mock is a negative control immunoprecipitate. Right: V5 antibodies for the recombinant IQGAP1 constructs expressed in COS7 cells shown in C were used for IP, and western blots (WB) were probed with antibodies for SEC8, EXO70 and SEPT2. Cells expressing the vector alone were included as negative controls (last lane). (C) Pull-down of the recombinant IQGAP1 proteins with GST-SEPT2 or GST-EXO70. Left: expression of the IQGAP1 constructs resolved on a 10-20% gradient SDS-PAGE and blotted with V5 antibodies. Right: the lysates shown on the left were incubated with immobilized GST alone (lysate from N1 cells is represented in the first lane), GST-SEPT2 (upper panel) or GST-EXO70 (lower panel) and processed as described in the Materials and Methods. The blots were probed with V5 antibodies to detect the recombinant IQGAP1 proteins. The bottom section of each panel demonstrates equal input of GST-SEPT2 (upper panel) or GST-EXO70 (lower panel). (D) Pull-down of GST-SEPT2 and GST-EXO70 with 6xHis-IQGAP1-N. Proteins from cells expressing V5-His-IQGAP1-N or V5-His vector alone were bound to the TALON cobalt resin, incubated with the bacterial lysates of GST-SEPT2 (upper panel) or GST-EXO70 (middle panel) and blotted with SEPT2 or EXO70 antibodies, respectively. 5% of the purified IQGAP1-N or bacterial lysate of GST-EXO70 or GST-SEPT2 were blotted in the lower panel. Data represent three independent experiments with identical results.