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. 2020 Feb 12;12(2):430. doi: 10.3390/cancers12020430

Figure 4.

Figure 4

Functional characterization of BRAF p.G593D, BRAF p.A598T, BRAF p.S607F, and BRAF p.S607P compared to BRAF wild type (WT), BRAF p.V600E, BRAF p.K601E, and empty vector (EMPTY). (a) Effect of BRAF mutation on the MAPK signaling pathway: representative Western Blot of ERK, p-ERK, BRAF, and p-BRAF proteins in HEK-293 cells transiently transfected with wild type or mutant BRAF plasmids. Vinculin was used as a loading control. (b) Effect of BRAF mutation on ERK1/2 phosphorylation: densitometric analysis of Western Blots showing the p-ERK/ERK ratio in HEK-293 cells transiently transfected with wild type and mutant BRAF plasmids. HEK-293 cells transfected with BRAF-V600E and BRAF-K601E show increased activation of the MAPK pathway compared to BRAF WT, whereas HEK-293 cells transfected with BRAF-G593D, BRAF-A598T, BRAF-S607F, and BRAF-S607P show similar MAPK activation to BRAF WT. Data have been normalized to nontransfected controls (not shown); data are reported as the mean ± SEM of at least two independent experiments. **: p < 0.005 and ***: p < 0.0001. (c) Effect of BRAF mutation on BRAF phosphorylation: densitometric analysis of Western Blots showing the p-BRAF/BRAF ratio in HEK-293 cells transiently transfected with wild type and mutant BRAF plasmids. There are no significant differences in BRAF phosphorylation levels between the different constructs. Data have been normalized to nontransfected control; data are reported as the mean ± SEM of at least two independent experiments.