Journal: British Journal of Cancer

Article Title: NFAT signalling is a novel target of oncogenic BRAF in metastatic melanoma

doi: 10.1038/sj.bjc.6605277

Figure Lengend Snippet: Nuclear factor of activated T-cells (NFAT) transcriptional activity, mRNA expression and protein expression in melanoma cell lines. ( A ) NFAT transcriptional activity measured in three human metastatic melanoma cell lines. BRAF mutational status is indicated for reference. Values are means from four independent experiments carried out in triplicate (± s.e.m.). ( B ) NFAT mRNA expression in primary melanocytes and in three metastatic melanoma cell lines as determined by real-time qPCR. Values are mean 2 − Δ Ct from RNA derived from two independent cultures (± s.d.). ( C ) Western blots indicate the expression of three previously described NFAT2 isoforms, NFAT3 protein and NFAT4 protein. 1°=primary melanocytes, CHL=CHL-1 cells, WM=WM266-4 cells. Equal protein loading was confirmed by blotting for β -actin. Statistical analysis in ( A ) performed by one-way ANOVA, * P ⩽0.05, ** P ⩽0.01, *** P ⩽0.001.

Article Snippet: Small interfering RNA (siRNA) targeting NFAT2 (cat. no. SI03057684) and NFAT4 (cat. no. SI03114118) (Qiagen, Crawley, UK) was transfected at a final concentration of 40 n M using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Activity Assay, Expressing, Derivative Assay, Western Blot

Journal: British Journal of Cancer

Article Title: NFAT signalling is a novel target of oncogenic BRAF in metastatic melanoma

doi: 10.1038/sj.bjc.6605277

Figure Lengend Snippet: Small interfering RNA (siRNA)-mediated knockdown of nuclear factor of activated T-cells (NFAT) 2 or NFAT4 reduces cyclooxygenase-2 (COX-2) protein induction. WM266-4 and A375 cells were transfected with siRNA targeting either NFAT2 ( A ) or NFAT4 ( B ). 48 h post-transfection, cells were treated with TPA (50 n M ) and ionomycin (1 μ M ) (T/I). UT=untreated control sample. At 4 h after treatment, cell lysates were prepared for western blotting. Membranes were probed with antibodies to detect NFAT2 ( A ), NFAT4 ( B ) and COX-2. Equal protein loading was confirmed by blotting for β -actin.

Article Snippet: Small interfering RNA (siRNA) targeting NFAT2 (cat. no. SI03057684) and NFAT4 (cat. no. SI03114118) (Qiagen, Crawley, UK) was transfected at a final concentration of 40 n M using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Techniques: Small Interfering RNA, Knockdown, Transfection, Control, Western Blot