Figure 3: A-Rafshort inhibits Ras-ERK signalling.
(A) Numbers of HeLa, GHD-1, and HCT-116 cells were determined in a time kinetic following overexpresion of A-Rafshort or A-RafWT. Results represent the mean with standard deviation (SD) of three independent experiments. (B) A-Rafshort inhibits phosphorylation of ERK. Hela cells were transfected with increasing amounts of A-Rafshort or A-RafWT. Cells were serum-starved for 16 hours (0.1% FCS) or serum-stimulated (10% FCS) for 5min after starvation as indicated. Lysates were immunoblotted for expression of the transfected flag-tagged Raf isoforms, phopho-ERK1/2, and ERK1/2 as loading control. (C) A-Rafshort inhibits ERK phosphorylation without affecting AKT phosphorylation. Hela cells were transfected with either A-Rafshort or A-RafWT. Cells were serum-starved for 16 hours (0.1% FCS) or serum-stimulated (10% FCS) for 5min after starvation as indicated. Lysates were immunoblotted for expression of the transfected flag-tagged Raf isoforms, phopho-ERK1/2, ERK1/2, phospho-AKT, and AKT. (D) A-Rafshort siRNA#3 specifically downregulates the A-Rafshort isoform and increases ERK phosphorylation. (left panel) Hela cells were transfected with four different siRNAs for A-Rafshort or A-RafWT. Cells were serum-starved for 16 hours and serum-stimulated for 5min. Lysates were immunoblotted for expression of A-Rafshort, A-RafWT, phopho-ERK1/2, and ERK1/2. Shown are the representative results from two independent experiments. (right panel) Hela cells were transfected with either A-Rafshort siRNA#3 or control siRNA. Cells were serum-starved for 16 hours (0.1% FCS) or serum-stimulated (10% FCS) for 5min after starvation as indicated. Lysates were immunoblotted for expression of A-Rafshort, phopho-ERK1/2, and ERK1/2.