Fig. 8. Depolarization-induced tyrosine phosphorylation of PYK2 is prevented by a dominant negative form of calcineurin. (A) PC12 cells transfected with GFP-PYK2 in the absence or presence of Flag-PD-CnA (phosphatase-dead calcineurin A) were treated with a depolarizing (High K⁺ 40 mM; +) or control (–) solution for 3 minutes. PYK2 tyrosine phosphorylation was analyzed by immunoblotting with an anti-P-Tyr antibody (pY, upper panel) and with an anti-PYK2 antibody (middle panel). The presence of Flag-PD-CnA was detected by Flag immunoblotting (lower panel). (B) Quantification of pY-PYK2 in A. Values are means ± s.e.m., of six cultures per group in two independent experiments. Two-way ANOVA: interaction between depolarization and PD-CnA co-transfection F_(1,16)=5.3, P<0.05, depolarization effect F_(1,16)=8.5, P<0.05, PD-CnA co-transfection effect F_(1,16)=5.5, P<0.01. Newman-Keuls test: control vs K⁺, ^**P<0.01, PD-CnA K⁺ vs K⁺, °°°P<0.001.