The IQ domain of IQGAP1 interacts with CaM in endothelial cells. (A) Schematic showing the two IQGAP1 domain truncation constructs fused to GFP. GRD, RasGAP-related domain; IQ, IQ motifs domain; RGCT, RasGAP C-terminus; WW, tandem tryptophan-containing domain. (B) The GFP-tagged IQGAP1 domain truncation constructs shown in A were transduced into iHUVECs grown on 60-mm tissue culture plates. After 2 d of expression, the cells were lysed in either the presence of CaCl₂ or EGTA. The protein lysates were then incubated with CaM Sepharose beads, and the beads were then eluted in 6× Laemmli buffer to be probed using Western blotting as described in the Materials and methods. The blot shown here was probed for IQGAP1. Both endogenous (Endog.) full-length IQGAP1 and construct Δ5,6 interact with CaM. However, construct Δ4–6, which lacks the IQ domain, cannot bind CaM in either the presence or the absence of calcium. Three separate experiments were performed. MW, molecular weight.