Fig. 3. Depolarization induces Ca²⁺-dependent nuclear translocation of PYK2 in hippocampal neurons in culture. (A) Rat hippocampal neurons in culture treated with high [K⁺]_o (High K⁺, 40 mM) or with a control solution for 3 minutes in the presence or absence of EGTA (5 mM, added 5 minutes before depolarization) and stained with an anti-PYK2 antibody were analyzed by fluorescence microscopy. Bars, 5 µm. (B) Quantification of the percentage of cells with nuclear PYK2. Values are means ± s.e.m. (six coverslips per group). Two-way ANOVA: interaction between depolarization and EGTA treatment F_(1,20)=48.35, P<0.0001, depolarization effect F_(3,20)=62.12, P<0.0001, EGTA treatment effect F_(1,20)=77.28, P<0.0001. Newman-Keuls test: PYK2 control vs K⁺, ^***P<0.001, PYK2 K⁺ vs (PYK2 + EGTA K⁺), °°°P<0.001. (C) Rat hippocampal neurons in culture treated with high [K⁺]_o (High K⁺, 40 mM) or with a control solution for 3 minutes and stained with an anti-PYK2 antibody were analyzed by confocal microscopy. A single optical section is shown. Bars, 5 µm.