CN and NFAT activity is increased in RCAN1 mutant cell lines. (A) HEK293 cells were transfected with constructs containing PPP3CA (CN) and either WT RCAN1 or the p.I162T RCAN1 variant. At 48 hours after transfection, the RCAN1 p.I162T variant–expressing cells displayed increased CN activity (P<0.001, n=12, t test) and (B) NFAT expression (P<0.001, n=6, t test) compared with WT RCAN1–expressing cells. For the NFAT luciferase assay, cells were also transfected with constructs to allow for both Firefly and Renilla dual-luminescence detection.