Extended Data Fig. 5: CIP2A interacts with TOPBP1.
a, Co-immunoprecipitation of CIP2A with TOPBP1. Whole-cell extracts from U2OS parental (WT) or MDC1-/- (KO) cells were subjected to immunoprecipitation with normal mouse IgG or a CIP2A antibody and were then immunoblotted with the indicated antibodies. Representative of two independent experiments. b, Micrographs of the LacR/LacO assay assessing the interaction between endogenous CIP2A and TOPBP1 variants fused to FLAG-LacR shown in Fig. 5f. Representative of 3 independent immunostainings. Scale bar = 10 µm. c, LacR/LacO assay assessing the interaction between endogenous CIP2A and TOPBP1 variants fused to FLAG-LacR. Data presented as the mean values ± S.D. n = 3 independent immunostainings; analyzed with one-way ANOVA, followed by multiple comparisons Dunnet test, all compared to FLAG-LacR; ∗∗∗p = 1 × 10−15. d, Alanine scanning of TOPBP1 (830-851) residues by yeast two-hybrid assay with CIP2A (1-560). Five residues that abolish the TOPBP1-CIP2A interaction when mutated to alanine were identified. AD, activation domain; BD, Gal4 DNA binding domain. Expression of proteins was verified by immunblotting but not shown. Representative of n = 2 independent sets of transformations. e, CIP2A and TOPBP1 interact directly. Upper: 1 μg of GST or GST-CIP2A (1-560) were separated by SDS-PAGE and stained with Coomassie. Lower blot: GST pull-down experiment with either GST or GST-CIP2A (1-560) incubated with either MBP, MBP-TOPBP1756–999 or MBP-TOPBP1756–999-3A. Bound proteins were processed for immunoblotting with an anti-MBP antibody. The CIP2A/TOPBP1 interaction has been observed > 5 times; the loss of interaction by the 3 A mutation is representative of two independent pulldown assays. Full blots are provided in Blot Source Data ED Fig. 5. Statistical analyses are found in Source Data ED Fig. 5.
