Fig. 1. Expression of 3xFLAG-hCaM and its binding to IQ(1-2-3-4) motif of IQGAP1. (A) Immunoblotting of endogenous CaM or overexpressed 3x FLAG-hCaM in HEK293T cells. (B) Colocalization of 3XFLAG-hCaM with IQGAP1 in HeLa cells. Immunocytochemistry analysis was performed to detect endogenous IQGAP1 and exogenous 3xFLAG-hCaM 24 h after transfection. Scale bar, 20 μm. (C) CaM binding of overexpressed IQ(1- 2-3-4) motif of IQGAP1 fused to EGFP in HEK293T cells using 3xFLAGhCaM IP or CaM 4B pull-down. EGFPIQ( 1-2-3-4) was equally bound to Ca²⁺/ CaM and apoCaM in both 3xFLAGhCaM IP and CaM 4B pull-down. Bound proteins were visualized by immunoblotting using an anti-GFP antibody. As a loading control, the amount of 3xFLAG-hCaM was detected using an anti-FLAG antibody.