Figure 4

Time lapse imaging of αCaMKII activation in SH-SY5Y 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 the application of ionomycin or KCl (50 mM)-application. Lower graph shows changes in relative 340 nm/380 nm ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 30–40 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 or KCl application. Lower graphs show changes in relative YFP/CFP emission ratio (open circle, ionomycin; filled circle, KCl). Black bar shows the period of ionomycin or KCl application. 11–25 cells were analyzed in each group. (D) Auto-phosphorylation at T286 of αCaMKII fusion proteins after ionomycin or KCI 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).