Fig 1: The anaphase promoting complex protects poly-ubiquitin signalling at DSBs.(a,b) Mobilization of APC12 (a) and Cdh1 (b) to sites of DNA damage. U2OS cells were transfected with GFP-APC12 or GFP-Cdh1 expression plasmids and the indicated siRNAs, microirradiated with laser and imaged at different time points. (c) Interaction of Cdh1 with Emi1 in S/G2 cells. HeLa cells stably expressing strep-tagged Cdh1 were synchronized with double thymidine, released into S/G2, exposed to 10 Gy IR and collected after 1 h recovery for immunoprecipitation analysis. (d) The accumulation of K11-linked poly-ubiquitin at sites of DNA damage. U2OS cells were transfected with ubiquitin (Ub) constructs, microirradiated and processed 30 min later for immunostaining of His tag and BRCA1. (e) The accumulation of the endogenous K11-linked Ub chains at sites of DNA damage. Immunostaining of K11-linked Ub and ?H2AX was performed 30 min after microirradiation. Top: representative images of K11-linked poly-(Ub) and ?H2AX immunostaining in U2OS cells transfected with the indicated siRNAs. Bottom: quantification of K11 linkage intensity at microirradiated regions (>110 cells quantificated). Error bars indicate SEM from three independent experiments. ** (in e) indicates P<0.001 (student’s t-test). Scale bars represent 10 µm.