PLCG1 mutants found in CTCL activate calcineurin/NFAT signaling. (A) Sanger DNA sequences corresponding to the human PLCG1 wild-type construct (upper) and the generated mutant -S345F and -S520F counterparts (lower). (B) Western blot from total HEK293T-cell lysates using anti-Myc (Covance) and anti-tubulin. Cells were grown in 6-well plates and transfected with PLCG1-Myc-WT (WT), PLCG1-Myc-S345F (S345F), and PLCG1-Myc-S520F (S520F) (1 µg each). The figure shows a representative experiment. (C) Luciferase assay in HEK293T cells grown in 24-well plates and cotransfected with NFAT-Luc (0.1 µg)/pRL-Null (0.05 µg), together with the indicated amount of each PLCG1 DNA construct. Before analysis, cells were starved overnight. *P < .05; **P < .01; ***P < .001. (D) HEK293T cells were handled as described earlier but use the best conditions for each PLCG1 DNA construct. Cells were serum-starved and incubated with the indicated amount of the inhibitors FK-506 and PLCi for 6 hours. N = 3; error bars indicate SEM.