Fig. 1. BRCA2 VUS alter PLK1 phosphorylation of BRCA2_1-250.

a PLK1 in vitro kinase assay with BRCA2_1–250. Top: The polypeptides encompassing 2×-MBP-BRCA2_1–250 WT or S193A, M192T, S196N, S206C, T207A mutations or the 2XMBP tag were incubated with recombinant PLK1 in the presence of γ³²P-ATP. The samples were resolved on 7.5% SDS-PAGE and the ³²P-labeled products were detected by autoradiography. Bottom: 7.5% SDS-PAGE showing the input of purified 2xMBP-BRCA2_1–250 WT and mutated proteins (0.5 μg) used in the reaction as indicated. Mr; molecular weight markers. b Quantification of the relative phosphorylation in (a). Data in (b) are represented as mean ± SD from four independent experiments. c PLK1 in vitro kinase assay performed as in (a) with recombinant PLK1 or the PLK1 kinase dead K82R mutant (PLK1-KD) together with BRCA2_1-250 WT as substrate, in the presence or absence of the PLK1 inhibitor BI2536 (50 nM) in the kinase reaction buffer. Mr; molecular weight markers. d Quantification of the relative phosphorylation in (c). Data in (d) are represented as mean ± SD from three independent experiments. b, d One-way ANOVA test with Dunnett’s multiple comparisons test was used to calculate statistical significance of differences (the p-values show differences compared to WT (b) or PLK1 (d); ns (non-significant)). Source data are available as a Source Data file.