Fig. 4.

LysoPC-induced NADPH oxidase activation requires TRPC6. A and B: WT, TRPC6^−/−, or TRPC5^−/− MAECs were incubated with lysoPC (10 µM) for 15 min. A: phospho-p47^phox(Ser³⁴⁵) and total p47^phox were detected by immunoblot analysis. Actin served as loading control (n = 4). B: densitometry measurements of and phospho-p47^phox(Ser³⁴⁵) are represented in graphic form: ●, medium control with WT ECs; ■, lysoPC with WT ECs; ▲, medium control with TRPC6^−/− ECs; ▼, lysoPC with TRPC6^−/− ECs, ◆, medium control with TRPC5^−/− ECs; ○, lysoPC with TRPC5^−/− ECs. C and D: human ECs (EA.hy926) were transiently transfected with NsiRNA (20 nM) or TRPC6 siRNA (20 nM) for 24 h. siRNA was removed. C: at 24 h after siRNA removal, cells were lysed and TRPC6 was detected by immunoblot analysis. Actin served as loading control (n = 3). D: after siRNA removal, EA.hy926 cells were incubated with lysoPC (12.5 µM) for 15 min. Phosphorylation of p47^phox and total p47^phox were detected by immunoblot analysis. Actin served as loading control (n = 3). (In A and D, lines indicate lanes rearranged from the same gel. All bands are from the same gel.)