Figure 2.

CaMKII isoform-specific regulation of activity-dependent GKAP turnover. (a) Effect of isoform-specific CaMKII RNAi on activity-dependent turnover of endogenous GKAP. Cultured hippocampal culture neurons (14 DIV) were transfected with α RNAi + β-galactosidase (β-Gal) or β RNAi + β-Gal. After 1 d post-transfection, neurons were treated with either Bic (40 μM) or TTX (2 μM) for 24 h and examined for the changes in GKAP clusters (red) by immuno-fluorescence staining. Transfected neurons were identified by β-Gal immunofluorescence (green). (b) Quantification of CaMKII RNAi effect on activity-dependent turnover of endogenous GKAP, measured by the changes in the cluster density from non-transfected neighboring neurons (Non-txf) and RNAi-transfected neurons. n > 20 per each construct. *** P < 0.001. (c) Rescue of β RNAi by a chimeric α-CaMKII with actin association domain of β-CaMKII (α-CaMKII-AD). Hippocampal neurons were transfected with β RNAi + α-CaMKII (WT) or β RNAi + α-CaMKII-AD, treated with TTX for 24 h, and immunostained for the CaMKII (green) and GKAP (red). Schematic diagram of CaMKII constructs are shown above the representative images. (d) Quantification of β RNAi rescue by α-CaMKII-AD on GKAP cluster density. n > 20 per each construct. *** P < 0.001. (e) Activation of CaMKII isoforms in Bic or TTX-treated hippocampal neurons. Neurons were untreated (C) or treated with Bic (B; 40 μM) or TTX (T; 2 μM) for 12 or 24 h. Total protein extracts were examined for protein levels of autophosphorylated-CaMKII (p-CaMKII) or individual CaMKII isoform. Full-length blots are presented in Supplementary Figure 13. Quantification of relative phospho-α-CaMKII and phospho-β-CaMKII levels (n = 3). Scale bars, 5 μm. Error bars represent s.e.m.