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Figure 7


Fig. 7. Depolarization-induced phosphorylation and nuclear translocation of PYK2 are abolished by cyclosporin A (CsA) and FK506. (A) PC12 cells were treated with high [K+]o (High K+ 40 mM) or control solution for 3 minutes in the absence or presence of cyclosporin A (10 µM, added 30 minutes prior to depolarization). Endogenous PYK2 was immunoprecipitated and analyzed by immunoblotting with anti-P-Tyr antibody (pY, upper panel). Whole cell lysates were analyzed by immunoblotting with PYK2 antibody (lower panel). (B) Quantification of pY-PYK2 in A. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=65.8, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (CsA + K+) vs K+, °°°P<0.001. (C) PC12 cells transfected with GFP-PYK2 were treated as in A and PYK2 immunofluorescence was analyzed by fluorescence microscopy. Bars, 10 µm. (D) Quantification of results in C. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=18.8, P<0.05. Newman-Keuls test: control vs K+, *P<0.05, (CsA + K+) vs K+, °P<0.05. (E) PC12 cells were treated as in A, except that FK506 (FK506 1 µM, added 30 minutes prior to depolarization) was used instead of cyclosporin A. Endogenous PYK2 was immunoprecipitated and analyzed by immunoblotting with anti-P-Tyr antibody (pY, upper panel). Whole cell lysates were analyzed by immunoblotting for pY402-PYK2 (middle panel) or PYK2 (lower panel). (F) Quantification of pY-PYK2 in E. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=153.1, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001. (G) Quantification of pY402-PYK2 in E. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=162.2, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001. (H) PC12 cells transfected with GFP-PYK2 were treated as in E and PYK2 immunofluorescence was analyzed by fluorescence microscopy. Bars, 10 µm. (I) Quantification of results in H. Values are means ± s.e.m. (three per group). One-way ANOVA: F(2,6)=166.2, P<0.0001. Newman-Keuls test: control vs K+, ***P<0.001, (FK506 + K+) vs K+, °°°P<0.001.





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