Fig. 4

Oncogenic BRAF enhances ERK5 expression and activation. a M26c melanoma cells and HEK-293T cells were transfected with equimolar amounts of pcDNA (control, −) or wt ERK5 in combination with pcDNA, constitutively active MEK5 (MEK5DD), wt BRAF, or BRAFV600E plasmids. Cells were lysed after 48 h and western blot was performed with the indicated antibodies. HSP90 was used as a loading control. b HEK-293T cells were transfected with equimolar amounts of pcDNA (control) or wt ERK5 in combination with pcDNA or BRAFV600E plasmids. Cells were treated with the ERK1/2 inhibitor SCH772984 (0.5 µM) and/or the CDK1 inhibitor RO-3306 (9 μM) during the last 18 h of transfection. Cells were lysed after 24 h and western blot was performed with the indicated antibodies. HSP90 was used as a loading control. c In vitro kinase assay for ERK5 immunoprecipitated from M26c cells transfected with equimolar amounts of pCAG, constitutively active MEK5 (MEK5DD) or BRAFV600E plasmids. MBP was used as a loading control. d In vitro kinase assay for ERK5 immunoprecipitated from A375 or SK-Mel-5 cells treated with 1 µM vemurafenib (Vem) or DMSO (Control) for 24 h. MBP was used as a loading control. Blots are representative images from five (a) or three (b) independent experiments. c–d Relative ERK5 kinase activity determined by densitometric quantification of pMBP normalized for MBP from three independent experiments (mean ± SD). pCAG and DMSO controls were set to 1. *p < 0.05 as determined by Student's t-test