Figure 3. CaMKII is involved in synaptic NMDAR-dependent ERK1/2 activation. A, Top, Immunoblots of p-ERK1/2 and ERK1/2 from extract of neurons exposed to 30 min of Bic/4AP (Syn), with or without STO-609 (5 μm, N = 7), KN-93 (5 μm, N = 5) and CN21a (5 μm, N = 4). Bottom, Mean (±SEM, N = 4–6) levels of p-ERK quantified from blots as above and normalized to control (open bars). Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test. *,#p < 0.05, significant difference compared with Bic/4AP and with control condition, respectively. B, Top, Developmental changes of GluN2A, GluN2B and α/βCaMKII, as measured by immunoblots from extracts of cortical neurons (DIV 7, 14, or 18) performed with an anti-α/βCaMKII, anti-GluN2B and anti-GluN2A. Bottom left, Mean (±SEM, N = 3) levels of α and βCaMKII expression quantified from blots as above.*p < 0.05, significant difference compared with 7 DIV. Bottom right, Mean (±SEM, N = 3) p-ERK1/2 levels quantified from immunoblots from neuronal cultures of the indicated DIV exposed to 30 min Bic/4AP and normalized to control (dotted line). *p < 0.05, significant difference compared with Bic/4AP at 7 DIV. Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test. C, Left, Representative images of neurons transfected with GFP ± αCaMKII shRNA, βCaMKII shRNA, or with αCaMKII shRNA + rescue mGFP-αCaMKII, and immunostained for p-ERKs (red), after 30 min of Bic/4AP incubation (N = 3; n = 32–45). Middle, Mean (±SEM) levels of p-ERK quantified from images as in left and normalized to control. Statistical analysis was performed by ANOVA followed by Bonferroni-Dunn's test. *p < 0.05, significant difference from the fluorescence intensity of GFP-transfected cells after synaptic activation. Right, Representative immunoblot of CaMKII, p-ERK1/2, and ERK1/2 from extracts of cortical cultures infected with a lentiviral αCaMKII shRNA and exposed or not to 30 min of Bic/4AP (Syn).