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. 2012 Sep 19;287(45):38367–38378. doi: 10.1074/jbc.M112.377499

FIGURE 2.

FIGURE 2.

The IQGAP1/RhoA interaction requires GTP binding and prenylation of RhoA. A, 293T cells were co-transfected with Myc epitope-tagged IQGAP1 and Flag-tagged RhoA constructs encoding wild type (Wt), constitutively active (V14 or L63), dominant-negative (N19) RhoA proteins, or empty vector. Anti-Flag immunoprecipitates were analyzed by Western blotting with anti-Myc antibody for the presence of IQGAP1 (top panel), or with anti-Flag antibody for the amounts of RhoA (bottom panel). Input lysates (2.5%) are shown in the right lanes. B, cells were transfected with IQGAP1 and the Rho constructs described in panel A; absolute amounts of GTP bound to anti-Flag immunoprecipitates were measured using a luciferase-based assay, as described under “Experimental Procedures.” C and D, cells were transfected with IQGAP1 and Flag-tagged wild type RhoA as in panel A, but cells were serum-deprived for 8 h and treated with EGF (20 ng/ml) for 5 min. In panel C, anti-Flag immunoprecipitates were analyzed by Western blotting for the presence of IQGAP1, and in panel D, GTP bound to anti-Flag immunoprecipitates was measured enzymatically as in panel B. E and F, 293T cells were co-transfected with Myc-tagged IQGAP1 and vectors encoding Flag-tagged RhoA(V14) in its normal prenylated form (C190 is the cysteine modified by geranyl-geranylation), or with a mutation in the prenylation signal (S190); anti-Flag immunoprecipitates were probed for the association of IQGAP1 (E) or for the amount of GTP bound to RhoA(V14/C190) and RhoA(V14/S190) (F).