Figure 5. A fraction of BRAF co-localizes with PAX3 in nuclei of muscle cells.

(A) Western blot analysis of cytoplasmic (C) and nuclear fractions (N) of C2C12 cells transfected with CA Braf (V600E), WT Braf, HA-Pax3 or HA-Pax3-GFP. n = 3. Successful fractionation was monitored by cytoplasmic GAPDH and the nuclear protein LAMIN A/C. Some cultures were treated with Dynasore (Dyna) for 30 min before fractionation as indicated. (B) Western blot analysis of immunoprecipitations of nuclear fractions isolated from migrating C2C12 cells after transfection with WT Braf, CA Braf (V600E), or HA-Pax3. n = 3. (C) High resolution confocal images of C2C12 cells transfected with WT, CA Braf (V600E) and HA-Pax3-GFP. (D) High resolution confocal image of a PAX3 and BRAF positive forelimb muscle precursor cell at E10.5 (left panel). A lower magnification is shown in the right panel.

DOI: http://dx.doi.org/10.7554/eLife.18351.009

Figure 5—figure supplement 1. Intracellular localization of BRAF in C2C12 cells.

(A) Immunofluorescence images of C2C12 cells in a Boyden micro chemotaxis chamber. Cultures were either not stimulated (Con) or treated with HGF after transfection with CA Braf (V600E). Note: Overlay of red BRAF and green PAX3 results in a yellow color of the nuclei. (B) High resolution confocal images of nuclei of migrating C2C12 cells transfected with WT and CA Braf (V600E). Cells were co-stained for P-BRAF and EEA1 as well as for P-BRAF and PAX3. Nuclei were visualized by DAPI. Note the absence of the endosomal marker EEA1 in nuclei, while P-BRAF is clearly visible within nuclei.

DOI: http://dx.doi.org/10.7554/eLife.18351.010