Figure 2.

BRAF^V600E activates nuclear factor of activated T-cells (NFAT) through a extracellular signal-regulated kinase kinase (MEK)-dependent pathway. (A) NFAT transcriptional activity was measured 16 h after treatment with the MEK inhibitor PD98059 (10 μM). Values are means from three independent experiments carried out in triplicate (±s.e.m.). Identically treated samples were prepared for western blotting and membranes were probed for phosphorylated extracellular signal-regulated kinase (ERK) (pERK 1/2) and total ERK (ERK 1/2). (B) The effect of BRAF^WT or BRAF^V600E overexpression on NFAT transcriptional activity in CHL-1 cells was measured by co-transfecting cells with an NFAT luciferase reporter±vectors expressing BRAF^WT or BRAF^V600E. BRAF^V600E-expressing cells were also treated with PD98059 where indicated. Untreated cells were transfected with NFAT reporter plus transfection reagent only and NFAT luciferase activity was measured 48 h later. Values are normalised to the untreated control and are means from four independent experiments carried out in triplicate (±s.e.m.). Identically treated samples were prepared for western blotting to confirm BRAF^WT and BRAF^V600E overexpression and downstream ERK phosphorylation. Equal protein loading was confirmed by blotting for GAPDH. Statistical analysis performed by t-test (A) or one-way ANOVA (B), ^** P⩽0.01, ^*** P⩽0.001 vs vehicle control. (C) Schematic of BRAF^V600E-induced NFAT activation.