Figure 4

Withdrawal of BRAF inhibitors from BRAF inhibitor-resistant cells leads to enhanced MAPK signaling. (a) Melanoma cells expressing the indicated genes and maintained in 1 μm vemurafenib for 2 weeks were then either maintained for a further 72 h (+) or withdrawn (−) from vemurafenib. The expression and phosphorylation of MAPK family members (RSK, ERK and MEK), AKT, protein translation-related proteins S6 ribosomal protein (S6 RP), ELK, eukaryotic translation initiation factor 4B (eIF4B), apoptosis marker (cleaved poly ADP ribose polymerase) and Fos-related antigen 1 (FRA1) in response to drug withdrawal was detected by immunoblotting. (b) Melanoma cells treated with increasing (0, 1 and 2.5 μm) concentrations of vemurafenib. After 24 h, the expression and phosphorylation of ERK-related proteins including dual specificity phosphatase 6 (DUSP6) was detected by western blot.