FIGURE 1.

Mutant TRPC6 induces ERK activation. A, parental M1R cells or subclones expressing the indicated FLAG-TRPC6 construct under a tetracycline-inducible promoter were treated with or without tetracycline for 16–24 h, as indicated, before lysis. Whole lysate blots were probed for FLAG, phosphorylated ERK1/2 (P-Erk), and total ERK1/2 as indicated. B, average normalized ratio of phosphorylated ERK1/2 to total ERK signal in different stable cell lines without or with tetracycline induction. One-way ANOVA with Tukey's multiple comparison test was used; *, p < 0.001 versus all other groups; n = 5. C, phosphorylated ERK, total ERK, and FLAG-TRPC6 levels in M1R cells transiently transfected with the indicated FLAG-TRPC6 constructs. D, whole cell lysates from differentiated murine podocytes transfected with the indicated HA-TRPC6 expression constructs or control vector were blotted for the expression of HA-TRPC6, phosphorylated ERK1/2, total ERK, and β-actin, as indicated. E, average normalized ratio of phosphorylated ERK1/2 to total ERK signal in podocytes expressing the indicated HA-TRPC6 protein. One-way ANOVA with Tukey's multiple comparison test; n.s., not statistically significant; n = 7. F, immunofluorescence of inducible M1R subclones expressing wild-type or R895C mutant FLAG-TRPC6 treated without (−) or with (+) tetracycline as indicated. Images were taken under identical exposure conditions. Error bars, S.E.