Fig. 3.

Effect of Src kinase, PLC, and PLA₂ on PTH-stimulated Na⁺-K⁺-ATPase α₁-subunit phosphorylation. OK cells were treated for 15 min with 100 nM PTH in the presence or absence of the Src kinase inhibitor PP2 (100 nM; A), the PLC inhibitor ET (30 μM; B), the PLA₂ inhibitors BEL (1 μM) or MAFP (1 μM) (C), or the calcium inhibitors BAPTA-AM (20 μM) or SKF-96365 (25 μM) (D). Cells were lysed, and crude membranes were prepared as described in methods. Na⁺-K⁺-ATPase α₁-subunit was immunoprecipitated (IP) using anti-Na⁺-K⁺-ATPase α₁-subunit antibodies as described in methods. Immunoprecipitated proteins were subjected to 10% SDS-PAGE, transferred, and probed with anti-phosphoserine antibodies. Nitrocellulose membranes were stripped and reprobed with anti-Na⁺-K⁺-ATPase α₁-subunit antibodies (data not shown). A representative Western blot is shown. Bar diagrams show arbitrary densitometry units of phosphorylated Na⁺-K⁺-ATPase α₁-subunit as means ± SE (n = 3). *P < 0.05 by ANOVA followed by Bonferroni analysis.