53BP1 foci formation at sites of CSR is RNF8 dependent. (A) RNF8^+/+, RNF8^−/−, and 53BP1^−/− B cells were stimulated with LPS and IL-4 for 3 d, left untreated (no IR), or exposed to 1 Gy of γ irradiation (IR) and then incubated for 30 min at 37°C before staining for 53BP1 (green). Bar, 5 µm. One representative out of two independent experiments is shown. (B) Distribution of 53BP1 in LPS/IL-4–activated B cells from RNF8^+/+ and RNF8^−/− mice. B cells were stained with anti-53BP1 antibodies followed by DNA FISH detection of the C_H region (left). The arrow indicates colocalization of 53BP1 and IgH. (right) The percentage of cells with colocalization of 53BP1 and IgH. A total of 52 cells positive for both signals were analyzed for each genotype in one experiment. Bar, 7 µm. (C and D) RNF8^−/− B cells stimulated with LPS/IL-4 were retrovirally infected with a WT or delRING form of RNF8 and stained for 53BP1 (C) or IgG1 (D) on day 3 (percentages are shown in D). One out of two representative experiments is shown. Bar, 7 µm.