Figure 6: Ets-1 activates the proximal NRAS promoter.

(a) Immunoblot for FLAG in BRAF mutant SK-Mel29 cells (express low endogenous Usp9x and Ets-1 levels) stably transfected with FLAG-Usp9x or FLAG-Ets-1 (top). Relative luciferase units (firefly/Renilla) in lysates from SK-Mel29 cells expressing (48 h) the proximal NRAS promoter, FLAG-Ets-1 or FLAG-Usp9x (bottom). (b) Immunoblot for FLAG in NRAS mutant WM1366 cells expressing FLAG-Ets-1 or FLAG-Usp9x (top). Relative luciferase units (firefly/Renilla) in lysates from WM1366 cells expressing the proximal NRAS promoter, FLAG-Ets-1 or full-length FLAG-Usp9x (bottom). (c) Proximal NRAS promoter sequence cloned from NRAS mutant SK-Mel147 cells, highlighting 5 putative ETS sites (designated E1M through E5M) derived from ChIP-SEQ analysis in other cell lines and visual inspection of the sequence. The consensus ETS binding sequence is highlighted below (boxed). (d) Relative luciferase units (firefly/Renilla) in lysates from SK-Mel29 cells expressing FLAG-Ets-1 and the proximal NRAS promoter (WT) or point mutants of each ETS putative binding site in the promoter region (E1M, E2M, E3M, E4M and E5M). (e) DNA-protein crosslinks from control and Usp9x KD cells were subjected to immunoprecipitation (as noted) before being used to prime a PCR reaction to detect the NRAS promoter. PCR products are shown (top) and compared with the input fraction (unfractionated DNA–protein complexes). Relative enrichment of the NRAS promoter for each condition is graphed below and represents the ave.±s.d. of three independent experiments.