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. 2016 Dec 1;5:e18351. doi: 10.7554/eLife.18351

Figure 4. BRAF directly interacts with PAX3 in migrating muscle precursor cells.

(A) PAX3, GST-PAX3 and PAX3 were immunoprecipitated from protein extracts of E10.5 wild type embryos or from C2C12 muscle cells after transfection with HA-Pax3 or CA Braf (V600E). Immunoprecipitations were analyzed by mass spectrometry after SDS-PAGE and in gel digestions. A selected list of proteins identified by Mascot search analysis is presented. (B) Analysis of the interaction of PAX3 and BRAF in E10.5 and E12.5 WT embryos by coupled immunoprecipitation/Western blot analysis. n = 3. (C) Western blot analysis of GST and GST-PAX3 immunoprecipitations after transfection of C2C12 cells with empty vector, Braf and CA Braf (V600E). n = 3. (D) Western blot analysis of PAX3 or BRAF immunoprecipitations from C2C12 transfected with CA Braf (V600E) after chemical cross-linking (SD) and cleavage of the cross-linker with DTT (SD+DTT). None = no cross-linker added. n = 3. (E) Western blot analysis of PAX3 immunoprecipitations from C2C12 transfected with WT Braf, CA Braf (V600E) and vector control (vector). n = 3.

DOI: http://dx.doi.org/10.7554/eLife.18351.007

Figure 4.

Figure 4—figure supplement 1. Selected lists of BRAF interaction partners identified by mass spectrometry analysis.

Figure 4—figure supplement 1.

Mass spectrometry was performed after in-gel digestion of Coomassie-stained bands from polyacrylamide gels. (A) Proteins identified after GST-PAX3 pull down of protein extracts from WT mouse embryonic forelimbs (E10.5). (B) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from extracts of WT mouse embryonic forelimbs (E10.5). (C) Proteins identified after immunoprecipitation of PAX3 form C2C12 myoblasts transfected with HA-tagged Pax3. (D) Proteins identified after immunoprecipitation of BRAF (antibody from BD Biosciences (612375)) from C2C12 myoblast cells after transfection with HA-tagged Pax3. Selected lists of proteins identified in the different co-immunoprecipitation experiments are shown.
DOI: http://dx.doi.org/10.7554/eLife.18351.008