JCS009631 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. PYK2 nuclear translocation is independent from Src-family kinases in PC12 cells. (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 PP2 (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(₁,₂,₈)=31.25, P<0.0005. Newman-Keuls test: control vs K⁺, ***P<0.001, (PP2 + K⁺) vs K⁺,°°°P<0.001. (C) PC12 cells were treated with high K⁺_o or with a control solution for 3 minutes in the presence of PP2 (10 μM, added 30 minutes prior to depolarization) or vehicle (DMSO). Endogenous PYK2 was detected with an anti-PYK2 antibody and nuclei were stained with DAPI. Bar, 10 μm. (D) Percentage of cells with nuclear PYK2 in C. Values are means ± s.e.m. (six coverslips per group). One-way ANOVA: F(₃,₂₀)=116.2, P<0.0001. Newman-Keuls test: control vs K⁺, ***P<0.001, (PP2 + K⁺) vs DMSO, <^b>⁺⁺⁺<^/b>P<0.001.

• Supplemental Figure S2 -

  Fig. S2. PYK2 KCl-induced phosphorylation and nuclear translocation are prevented by a calcineurin inhibitor in hippocampal slices. (A) Rat hippocampal slices were treated with vehicle (Control) or high K⁺_o (High K⁺) for 2 minutes in the absence or presence of a calcineurin inhibitor, FK506 (1 μM, applied 30 minutes before depolarization). PYK2 was immunoprecipitated and blotted with a P-Tyr antibody (pY, upper panel). Slice SDS lysates were blotted with an anti-PY402-PYK2 antibody (middle panel) or with an anti-PYK2 antibody (lower panel). (B) Quantification of the optic density of immunoreactive pY-PYK2 bands. Values are means ± s.e.m. (three to four slices per group). One-way ANOVA: F(₂,₉)=11.2, P<0.01. Newman-Keuls test: **P<0.01 compared with control,°P<0.05 compared with KCl. (C) Quantification of the optic density of immunoreactive bands in the P-Y402-PYK2 immunoblot, expressed as a percentage of controls. Values are means ± s.e.m. (n=3 per group). One-way ANOVA: F(₂,₆)=18.3, P<0.01. Newman-Keuls test: **P<0.01 compared with control,°°P<0.01 compared with KCl. (D) Hippocampal slices treated as in A were stained with an anti-PYK2 antibody. Bar, 50 μm. (E) Quantification of the percentage of cells with PYK2-positive nuclei. Values are means ± s.e.m. (five to seven slices per group). One-way ANOVA: F(₂,₁₅)=34.6, P<0.001. Newman-Keuls test: ***P<0.001 compared with control,°°°P<0.001 compared with KCl.