SNF8 effect on NFAT-mediated transcription. A. Cells stably expressing tetracycline-inducible wild-type, R895C or E897K TRPC6 were transfected with an NFAT-responsive luciferase reporter plasmid and either control plasmid or HA-SNF8 expression plasmid. Normalized luciferase activity was determined after TRPC6 expression was induced for 24 hours. Results are means ± SEM; one way ANOVA; * p<0.05 vs. control; ***p<0.001 vs. control. B. Western blots to confirm the expression of FLAG-TRPC6 and HA-SNF8 in lysates from cells used in the luciferase assays in (A). Actin was utilized as a loading control. C. Cells expressing wild-type or R895C TRPC6 under a tetracycline inducible promoter were transfected with plasmid encoding for either SNF8 shRNA (SNF8 Knockdown) or a scrambled shRNA (control), and luciferase reporter plasmids. 24 hours after transfection, TRPC6 expression was induced by the addition of tetracycline as indicated (induced). Cells cultured in the absence of tetracycline (uninduced) did not express detectable amounts of TRPC6, and were used as controls. 24 hours post-induction, luciferase reporter assays were performed. Results are means ± SEM; one way ANOVA, ***p<0.001 vs control shRNA. D. M1R cells were transfected with plasmids encoding for either an shRNA targeting SNF8, or a scrambled shRNA (control), as well as GFP. 48 hours post-transfection, cells were trypsinized and GFP expressing cells isolated by FACS cell sorting. Lysates from GFP positive cells were analyzed by Western blot for expression of SNF8. Actin expression was used as a loading control.