Interactions of mutant CaMs with SK2 channels revealed by coimmunoprecipitation assay. (A and B) Diagrams depicting the SK2 channel subunit with an HA tag in the S1-S2 extracellular loop and CaM with a FLAG tag in the C terminus under the cytomegalovirus (CMV) promoter. RBS, ribosome-binding site. (C) Coimmunoprecipitation using anti-Flag antibody (α-Flag) to pull down SK2 channels identified using anti–HA antibody (α-HA) in the presence of 2 mM EGTA. The right two lanes show the results from nontransfected HEK 293 cells (NT) and cells transfected with SK2 channels only (SK2) as negative controls. (D) Summary data of the findings in C. The background subtracted intensity for SK2 bands using anti–HA antibody was normalized to CaM bands using anti-Flag antibody (NIH ImageJ). Data shown represent mean ± SEM (n = 3). Statistical analyses were performed using one-way ANOVA combined with Tukey’s test; *, P < 0.05 for CaMWT compared with CaMF90L, CaMD96V, CaMN98S, and CaMD130G, and *, P < 0.05 for CaMF90L compared with CaMD96V, CaMN98S, and CaMD130G (the P values are as follows: CaMWT versus CaMF90L, 0.00003; CaMWT versus CaMD96V, 0.002; CaMWT versus CaMN98S, 0.004; CaMWT versus CaMD130G, 0.004; CaMF90L versus CaMD96V, 0.03; CaMF90L versus CaMN98S, 0.02; and CaMF90L versus CaMD130G, 0.02). IP, immunoprecipitation.
