Fig. 8.

Effect of 20-hydroxyeicosatetraenoic acid (20-HETE) and arachidonic acid on Na⁺-K⁺-ATPase regulation. OK cells were treated for 15 min with 10 μM 20-HETE or 5 μM arachidonic acid. A: cells were lysed, and Na⁺-K⁺-ATPase α₁-subunit (top) or PKCα (bottom) was immunoprecipitated from crude membranes. Immunoprecipitated proteins were subjected to 10% SDS-PAGE, transferred to nitrocellulose membrane, and analyzed by Western blotting using anti-PKCα or anti-Na⁺-K⁺-ATPase α₁-subunit antibodies. The blots were stripped and reprobed for total Na⁺-K⁺-ATPase α₁-subunit or PKCα immunoprecipitated. A representative blot is shown (n = 3). B: a sample from the Na⁺-K⁺-ATPase α₁-subunit immunoprecipitate was used to analyze for Na⁺-K⁺-ATPase α₁-subunit phosphorylation using anti-phosphoserine antibodies. The blots were stripped and reprobed for total Na⁺-K⁺-ATPase α₁-subunit immunoprecipitated. A representative blot is shown (n = 3). C: OK cells were treated for 15 min with 10 μM 20-HETE or 5 μM arachidonic acid. Cell surface proteins were biotinylated as described in methods. Biotinylated proteins were precipitated using streptavidin beads, separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and analyzed by Western blotting using anti-Na⁺-K⁺-ATPase α₁-subunit antibodies. A representative blot from 3 independent experiments is shown (n = 3). D: OK cells were treated for 15 min with 10 μM 20-HETE or 5 μM arachidonic acid in the presence or absence of 100 nM PKCα inhibitory peptide or 10 μM U0126. ⁸⁶Rb uptake was measured as described in methods. Bars represent ouabain-sensitive ⁸⁶Rb uptake as means ± SE from 6 individual experiments run in triplicate (n = 6). *P < 0.05 by ANOVA followed by Bonferroni analysis.