Fig. 2

A, B Western blot analysis of total and phosphorylated MAPKs in cultured HEK293 cells overexpressing the BRAF mutant allele. Vector: transfected with the empty vector; WT: transfected with wildtype BRAF construct; c.362C > A: transfected with the novel BRAF variant (NM_004333.6: c.362C > A, p.Arg121Thr); 735A > C: transfected with the positive control variant (NM_004333.6:c.735A > C, p.Leu245Phe), a known pathogenic variant associated with LEOPARD syndrome 3. The proportion of phosphorylated p38 MAPK increased significantly in the c.362C > A group (44.80% vs 24.38%, p-value = 0.0016), while did not change significantly in the positive control group (24.24% vs 24.38%). The proportion of phosphorylated ERK MAPK increased significantly in both c.362C > A group (57.71% vs 32.85%, p-value = 0.001) and the positive control group (57.28% vs 32.85%, p-value = 0.013), but did not differ significantly between the two mutant groups(p-value = 0.963). C Quantitative PCR analysis of male-sex-differentiation-related genes. The expression of SRY (p-value = 0.002), SOX9(p-value = 0.0003), and FGF9 (p-value = 0.007) decreased by 50%, 42%, and 41% respectively in the c.362C > A (p.Arg121Thr) group. While in the positive control group, no reduction in expression of these gene was observed