Figure 3
Time lapse imaging of αCaMKII activation in HeLa cells. (A) Changes in intracellular Ca2+ concentration measured using Fura2-AM. Upper panels show images of typical 340 nm/380 nm ratio at each time point before and after ionomycin application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, DMSO). Black bar shows the period of ionomycin application. 12–18 cells were analyzed in each group. (B, C) Changes in the interaction of YFP-αCaMKII with CFP-CaM (B) and the conformational change of YFP-αCaMKII-CFP (C). Upper panels show images of typical YFP/CFP emission ratio before and after ionomycin application (upper images, –KN-93; lower images, +KN-93). Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, ionomycin with KN-93). Black bar shows the period of ionomycin application. 9–13 cells were analyzed in each group. (D) Effect of removal of ionomycin by wash out at 0.5 or 1 min following ionomycin application. Upper graph shows the interaction of YFP-αCaMKII with CFP-CaM (open circle, –wash; triangle, 0.5 min; filled circle, 1 min). Lower graph shows the conformational change of YFP-αCaMKII-CFP (open circle, -wash; triangle, 0.5 min; filled circle, 1 min). 10–12 cells were analyzed in each group. (E) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin application at each time point (0, 1, 5 and 10 min). Upper panel shows Western blot analyses analyzing the expression level of phospho-αCaMKII fusion proteins and total-αCaMKII fusion proteins. Lower graph shows relative ratio of phospho-αCaMKII/αCaMKII levels (n = 3).