(A) Study protocol. Mice heterozygous for floxed BRAFV600E and hemizygous for Myh6-directed tamoxifen-inducible Cre were treated with 40 mg/kg (i.p.) tamoxifen (Tam) or vehicle (Veh). Echocardiograms (ECHO) were taken at baseline (BL) then at 10 d or 6 weeks after tamoxifen treatment. (B) Confirmation of recombination for BRAFV600E knock-in was based on introduction of an Xba1 restriction site in the mRNA following recombination. cDNA was prepared from RNA extracted from heart, liver or kidney, 24 h or 10 d after treatment with tamoxifen or vehicle. cDNA was PCR amplified and subject to Xba1 digestion, resulting in appearance of additional bands (arrows) in hearts (not liver or kidney) following treatment with tamoxifen, but not vehicle. (C) qPCR analysis of mRNA expression of RAF isoforms in samples of mouse hearts treated with vehicle or tamoxifen for 24 h (upper panel) or 10 d (lower panel). (D and E) Immunoblotting of RAF isoforms or Gapdh, in addition to phosphorylated (phospho-) or total MKK1/2, ERK1/2, p90RSK, Akt and p70S6K in samples of mouse hearts treated with vehicle or tamoxifen. Representative immunoblots are shown (positions of relative molecular mass markers in kDa are on the right of each blot) with densitometric analysis. (F and G) qPCR for mRNA expression in mouse hearts after tamoxifen-treatment for 24 h (F) or 10 d (G). Data are provided as individual values with means ± SEM. Statistical analysis: unpaired two-tailed t-test.