Fig. 2: BARD1 interacts with a conserved p50 phospho-serine motif.

a Putative BARD1 BRCT-binding motif. Motif in human p50 is underlined. Sequence of p50 mutants (lower). b 293T cells expressing FLAG-BARD1 and the indicated HA-p50 construct. IP and IB performed with the indicated antibodies. c 293T cells expressing TopBP1^ER or GFP^ER transfected with p50^wt (left) or p50^S337A (right) and treated with vehicle or TAM (4 h). IP with anti-HA and IB with anti-phospho-p50-S337 antibody. d 293T cells expressing TopBP1^ER were transfected with si-CHK1 or si-control and treated with vehicle or TAM. IB was performed with the indicated antibody. e Kinase assay with bacterially expressed p50^wt or the indicated p50 mutant and recombinant CHK1. Autoradiogram (³²P) and IB with indicated antibody of the same membrane. Arrow, phosphorylated p50. f Co-IP in primary WT MEFs expressing TopBP1^ER or GFP^ER treated with vehicle or TAM (4 h). IP with anti-p50 and IB with the indicated antibody. g WT MEFs expressing TopBP1^ER transfected with si-CHK1 were treated with TAM (4 h). IP with anti-p50 and IB with the indicated antibody. h Co-IP in WT MEFs following endogenous IP with anti-p50 and IB with anti-Bard1 or the indicated antibody. Lysates were treated with lambda phosphatase (λ PPase) or vehicle as indicated. i Co-IP in 293T cells expressing FLAG-BARD1 and the indicated HA-p50 construct. IP with anti-FLAG and IB with anti-HA antibody. j Nickel column purification of 6His-p50^wt or 6His-p50^DE2A incubated with GST-BRCT or GST alone. IB of eluted protein performed with anti-p50 antibody. k Co-IP in 293T cells transfected with empty vector (EV) or the indicated FLAG-BARD1 construct. IP was performed with anti-p50 and IB with anti-FLAG. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.