Figure 6. Nuclear translocation of BRAF and migration of muscle cells depend on intact endosomal trafficking.
(A) Western blot analysis of isolated subcellular fractions of C2C12 cells after transfection of CA Braf V600E and knockdown of Eea1 or Erk1/2. n = 3. Knockdown of Eea1 prevented accumulation of BRAF in the nucleus (N). C: cytoplasm. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. (B) Knock down of Braf (siBRAF) but not of Eea1 (siEea1), Dnm-2 (siDnm2), Cltc (siCltc), Cav1 (siCav1) and Arf6 (siArf6) did prevent phosphorylation of ERK1/2. Western blot analyses of siRNA transfected C2C12 cells are shown. n = 3. Actin served as loading control. siRNA knock-down efficiencies for Pax3 (85%), Braf (70%), Arf6 (80%), Cav1 (75%), Cltc (70%), Dnm2 (75%) and Eea1 (60%) were determined by Western blot analysis. (C) Immunofluorescence staining of migrating C2C12 cells after knockdown of Eea1 or Erk-1/2. Cultures were transfected with control vector, Braf or CA Braf (V600E) as indicated. Con indicates control cultures and siCon means control siRNA. siRNA knock-down efficiencies for Eea1 (60%) and Erk (95%) were determined by Western blot analysis. Cell numbers were determined by counting the number of DAPI-stained nuclei. A statistical analysis of siEea1 versus siCon in CA Braf (VE600E) transfected cultures is shown. n = 12; Mann-Whitney-U test, (p*<0.05).