Fig. 5

An interaction between ID3 and MDC1 is required for the recruitment of MDC1 at DSBs. a A schematic representation of wild-type ID3 (WT) and deletion constructs is shown. Domain boundaries, including the HLH region (25–81), are indicated using numbering for the human ID3 protein sequence. The relative affinity of MDC1 for the ID3 deletion mutants is indicated on the right side of the figure. b The HLH domain of ID3 is required for binding to MDC1. HEK293T cells were co-transfected with HA-tagged MDC1 and GFP-tagged deletion mutants of ID3, as indicated. After 48 h, cells were exposed to IR, and 3 h later, cell lysates were subjected to immunoprecipitation (IP) and immunoblotting (IB) using the indicated antibodies. * indicated heavy chain. c HEK293T cells were co-transfected with HA-MDC1 and one of three different versions of GFP-ID3: WT, T62A, or S65A. Cell lysates were subjected to IP using an anti-HA antibody and IB using antibodies indicated to the right of the image. d HEK293T cells were co-transfected with HA-MDC1 and one of three different versions of GFP-ID3: WT, S65A, or S65E. Cell lysates were subjected to IP using an anti-HA antibody and IB using antibodies indicated to the right of the image. e, f HeLa cells with a stable ID3 knockdown expressing HA-WT MDC1 were reconstituted with the indicated ID3 constructs or with a mock control. The exogenous ID3 and MDC1 were analyzed by IB using the indicated antibodies (e). Immunostaining for HA-MDC1 and GFP-ID3 was carried out 1 h after exposure to IR (f). Scale bars: 10 μm. The histogram in the lower panel quantifies the number of IR-induced foci (n = 3). g, h Similar to e, f but with the indicated point mutants in the HLH of ID3 expressed in stable ID3 knockdown HeLa cells (n = 3). Scale bars: 10 μm. Uncropped blots of this Figure accompanied by the location of molecular weight markers are shown in Supplementary Fig. 14. Data are presented as means ± s.d. P values are based on Student’s two-tailed t-test: **P < 0.01. ns, not significant