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Fig. 1. Depolarization induces nuclear translocation of PYK2 in hippocampal slices, independently of Src-family kinases. (A) Rat hippocampal slices incubated in physiological conditions were placed for 2 minutes in high [K+]o (High K+, 40 mM) or normal [K+]o (Control) and immediately fixed. Slices were cut into 30-µm thick sections and stained with anti-PYK2 antibody and DAPI. Bars, 50 µm. (B) Quantification of the percentage of cells with PYK2-positive nuclei. Values are means ± s.e.m., six slices per condition, Student's t-test: ***P<0.001. (C) Time course of the effects of high [K+]o on nuclear localization of PYK2 immunoreactivity. High K+ or control solution was added between 0 and 2 minutes (horizontal bar) and then replaced by standard ACSF. Slices were fixed at the indicated times. Values are means ± s.e.m., two to five slices per data point. Two-way ANOVA: interaction between time and treatment F(3,28)=18.3, P<0.001, treatment effect F(1,28)=29.5, P<0.001, time effect F(3,28)=15.4, P<0.001. Point by point comparison with controls using the Bonferroni test: ***P<0.001. (D) Rat hippocampal slices were treated with vehicle or high [K+]o for 2 minutes in the absence or presence of a Src-family inhibitor, PP2 (20 µM, added 30 minutes before depolarization). Slice homogenates were blotted with a phosphotyrosine antibody (pY, top panel) or with an anti-phospho-Tyr418-Src antibody (p-Src, bottom panel). The positions of PYK2 (110 kDa, p110-PYK2) and Src (60 kDa, p60Src) are indicated. (E) Rat hippocampal slices were treated as in D and PYK2 localization analyzed by immunofluorescence. Bar, 50 µm. (F) Quantification of the percentage of cells with PYK2-positive nuclei in slices treated as in E. Values are means ± s.e.m., five to eight slices per group, one-way ANOVA: F(2,17)=49.1, P<0.001. Newman-Keuls test: ***P<0.001 compared with control.
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