Western-blot analysis of ERK phosphorylation levels after sorafenib and RNAi treatment. (a) Sorafenib treatment leads to decreased levels of ERK phosphorylation. Cells were treated in serum free conditions with 4 uM of sorafenib or DMSO and proteins were extracted after 1 h, 12 h, 24 h and 48 h. The levels of phosphorylated ERKs after treatment were quantified relative to total ERKs and compared with the DMSO control. As shown in the graph, the levels of ERK phosphorylation decreased in all the cell lines at different time points, the levels were variable and transient in the time points analyzed in the different cell lines. (b) BRAF knockdown by RNAi induced decreased levels of ERK phosphorylation 48 h after transfection as detected by Western-blot analysis. Quantification of the levels of phosphorylated ERKs relative to total ERKs and compared with RNAi control (see graph), showed that BRAF-C2 siRNA inhibited ERK phosphorylation in 8505C and C643, but not in TPC1 cells. All values presented are the mean of at least three independent experiments.