Fig. 6. PYK2 redistribution in PC12 cells is independent of its autophosphorylation and kinase activity. (A) PC12 cells were transfected with wild-type GFP-PYK2 (WT, lanes 1, 2), GFP-PYK2 Y402F (Y402F, lanes 3, 4) or kinase-dead mutant K457A lanes (K457A, lanes 5, 6), and treated with control or depolarizing solution (K⁺ 40 mM) for 3 minutes. PYK2 was analyzed by immunoblotting (100 µg protein) with anti-pY402 antibody (upper panel) or with anti-PYK2 antibody (lower panel). (B) PC12 cells transfected with GFP-PYK2 WT, Y402F or K457A were treated with high [K⁺]_o or control solution for 3 minutes. PYK2 immunoreactivity was analyzed by fluorescence microscopy. Bar, 10 µm. (C) Quantification of the percentage of cells with nuclear GFP-PYK2. Values are means ± s.e.m. (four coverslips per group). Two-way ANOVA: interaction between mutation and depolarization F_(2,6)=2.1, P>0.05, depolarization effect F_(1,6)=72.7, P<0.0001, mutation effect F_(2,6)=1.1, P>0.05. Newman-Keuls test: WT control vs K⁺, ^**P<0.01, Y402F control vs K⁺, ^*P<0.05, K457A control vs K⁺, ^**P<0.01. (D) Homogenates (70 µg protein) from control or K⁺-treated PC12 cells were loaded on a 7% acrylamide gel. Note the downward shift induced by depolarization.