Activation of RAF and ERK1/2 signalling in cardiomyocytes by the type 1 RAF kinase inhibitor, SB590885.
(A–D) Superdex 200 FPLC separation of RAF complexes in unstimulated neonatal rat cardiomyocytes. Fractions were immunoblotted with antibodies to ARAF, or BRAF and RAF1. Representative blots of all column fractions (A) and comparing expression in fractions across different blots (B) are shown. (C) Column calibration with relative molecular mass (RMM) standards. (D) Elution profiles of ARAF, BRAF and RAF1. Results are means ± SEM (n = 3). (E) Immunoblots of extracts from neonatal rat cardiomyocytes exposed to 0.1 µM SB590885 or 0.1 µM endothelin-1 (ET-1) for the times indicated. Total extracts were immunoblotted for phosphorylated BRAF(S445) and RAF1(S338) (upper blot) or the total proteins (lower blot). Representative blots of four independent cardiomyocyte preparations are shown. (F) RAF kinase activities in neonatal rat cardiomyocytes exposed to the indicated concentrations of SB590885 or 0.1 µM ET-1 for the times shown. Activities were assayed using GST-MKK1 following immunoprecipitation (IP) of RAF1 or BRAF. Phosphorylation of GST-MKK1 was detected with antibodies to phosphorylated MKK1/2. Assay samples were also immunoblotted for total MKK1/2, BRAF and RAF1. Representative blots are shown (left panels) with densitometric quantification (right panels; results are the ratio of phosphorylated/total GST-MKK1, normalised to the mean of controls). Data are provided for each experiment with means ± SEM (n = 4). Statistical analysis used one-way ANOVA with Holm–Sidak's post-test. (G–J) Immunoblot analysis of phosphorylated (phospho-) and total ERK1/2 in extracts from neonatal rat cardiomyocytes (G,H and I) or Langendorff perfused adult rat hearts (J). Cells were exposed to the indicated concentrations of SB590885 for 20 min (G) or the indicated concentration of SB590885 for the times shown (H and I). Rat hearts were perfused (15 min) under control conditions or with 1 µM SB590885 (SB59) (J). Representative immunoblots are shown with densitometric analysis. Results of the cell experiments are provided as means ± SEM for n = 3 or 4 independent cell preparations as indicated. Heart perfusion data are provided as individual results with means ± SEM (n = 4). Statistical analysis used one-way ANOVA with Holm–Sidak's post-test and individual P values are provided. Positions of relative molecular mass markers (kDa) are on the right of the immunoblots.