*4.8. Western Blot*

Cells were lysed in RIPA with phosphatase inhibitor and protease inhibitor (Roche). Anti-phospho-NKCC1 antibody (Millipore, 1:5000), anti-phospho-SPAK antibody (Millipore, 1:4000), anti-phospho-eNOS antibody (Millipore, 1:8000), and anti-rabbit secondary antibody were used to detect phosphorate-NKCC1 and phosphorate-SPAK. The two most thoroughly studied sites of phospho-eNOS are the activation site Ser1177 and the inhibitory site Thr495. Several protein kinases, including Akt/PKB, PKA, and AMPK activate eNOS by phosphorylating Ser1177 in response to various stimuli. In our study, we used antiphospho-eNOS antibody (Ser1177). Sample loading was measured by β-actin (1:30,000) with anti-mouse as secondary antibody.
