Fig. 5: Ubiquitination of BARD1 by RNF19A is essential for HR regulation.

a HEK293T cells transfected with WT or NES deletion (ΔNES) mutant of Flag-BARD1 were lysed and pulled down by Ni-NTA and immunoblotted with indicated antibodies. b Indicated plasmids were transfected into HEK293T cells. Cell lysates were pulled down by Ni-NTA and immunoblotted as indicated. c WT or 2KR Flag-BARD1 were transfected into HEK293T cells. Cell lysates were immunoprecipitated with Flag beads and subjected to immunoblot with indicated antibodies. d Indicated plasmids were transfected into HEK293T cells. After 6 h MG132 treating, cell lysates were immunoprecipitated with Flag beads and subjected to immunoblot with indicated antibodies. e–g U2OS cells stably expressing Vec or RNF19A (exo) using plvx2-CMV-RNF19A (no tag) were further stably overexpressed with WT or 2KR Flag-BARD1. Each group of cells was transfected with BARD1 siRNAs for 48 h and treated with IR (2 Gy, 1 h). BRCA1 and Flag-BARD1 foci were detected by immunofluorescence (e). Quantification of focus signals per cell (n = 100) is shown in f: Flag-BARD1 and g BRCA1. Scale bars, 10 μM. h HEK293T cells stably expressing Vec or RNF19A (exo) were further stably overexpressed with WT or 2KR Flag-BARD1 and transfected with BARD1 siRNAs for 48 h and subjected to DR-GFP based HR assay. i The sensitivity of cells shown in (e) to Olaparib was assessed using colony formation assay. j U2OS ER-AsiSI cells stably expressing Vec or GFP-RNF19A were further stably overexpressed with WT or 2KR Flag-BARD1 and transfected with BARD1 siRNAs for 48 h and treated with 4-OHT for 4 h. Cells were digested and measured of DNA resection. k U2OS cells stably expressing Vec or GFP-RNF19A were further stably overexpressed with WT or 2KR Flag-BARD1 and treated with IR (10 Gy, 1 h). Nuclear (Nuc) and cytoplasmic (Cyto) proteins were extracted respectively. Cell lysates were immunoblotted with indicated antibodies. Error bars represent means ± s.d. of three (f, g, i, and j) or four (h) independent experiments. p values are determined by unpaired two-sided t test in f–j. Source data are provided as a Source Data file.