MDC1 and p53 interact following DNA damage through the tBRCT domain of MDC1.

<p>(a) A scheme of the different domains of p53. (b) A scheme of the different domains of MDC1. Note that in A, and B the images are not to scale as p53 is about 5 times smaller than MDC1. (c) 293T control cells transfected with empty vector or cells overexpressing HA-tagged p53 were either untreated (-) or treated with NCS (+). Following 1 hr incubation, the proteins were extracted and subjected to co-IP using anti-HA antibodies. Control for the co-IP was done with anti-GST antibodies. Detection was done using antibodies directed against endogenous MDC1 and phopsho-Ser15 of p53 (ph-p53). (d) The tBRCT domain of MDC1 directly interacts with p53. GST pull-down assay was performed with GST-p53 and His-tBRCT or His-FHA. All recombinant proteins were expressed in bacteria. Proteins were separated on a SDS gel and stained with Coomassie blue. (e) Endogenous p53 and MDC1 interact. Protein extracts prepared from 293 cells that were induced with 5 Gray of ionizing radiation and left for recovery for 1 hr were used in an IP experiment using antibodies directed against MDC1-tBRCT (α-BRCT) or against MDC1-FHA (α-FHA). Bound proteins were detected in Western blot using anti-p53 antibodies. Inputs present 5% of the extract used in the experiment.