Fig. 7

Identification and characterization of BRAF protein isoforms. a Schematic representation of the 3’ terminal region of reference, X1, and X2 BRAF mRNAs, as well as of the corresponding C-terminal regions of reference, X1, and X2 BRAF proteins. b Immunoprecipitation of BRAF protein in A375 cells. Endogenous BRAF was immunoprecipitated using a specific antibody that recognizes the N-terminal domain (IP-BRAF). As negative control, no antibody was used (No Ab). The basal level of BRAF in the cell lysate is shown in Input. c Identification by mass spectrometry of the C-terminal peptides of BRAF-ref and BRAF-X1. Immunoprecipitated BRAF was subjected to LC-MS analysis. The presence of both isoforms is revealed by the detection of isoform-specific peptides (in green). d Best transitions (BRAF-ref: 352 and 904; BRAF-X1: 1046 and 1117) of the two BRAF protein isoforms by mass spectrometry (MRM based method). e-f Upon the transient transfection of PIG-BRAFV600E-ref, X1, and X2 plasmids in HEK293T cells, western blot indicates that only reference and X1 BRAFV600E are efficiently translated and able to phosphorylate MEK, while X2 is not (e). This occurs in spite of the fact that according to real-time PCR for total BRAF levels, all 3 mRNAs are transcribed at similar levels (f). g-i Upon the stable infection of pMSCVHygro-Δ[3–10]BRAFV600E-ref, X1, and X2 plasmids in A375 cells, real-time PCR for total BRAF indicates that all 3 mRNAs are transcribed at similar levels (g), but western blot indicates that reference and X1 Δ[3–10]BRAFV600E are efficiently translated and able to phosphorylate MEK even in the presence of vemurafenib, while X2 is not (h). Consistently, only Δ[3–10]BRAFV600E-ref and -X1 are able to decrease the sensitivity of A375 cells to vemurafenib (i). The pictures are taken from 1 out of 3 independent experiments performed, all with comparable outcome. The graphs represent the mean ± SEM of 3 independent experiments