Figure 7.

RNF168 and 53BP1 are not limiting factors during immunoglobulin class switch recombination (CSR). (A) BRCA1^Δ11/Δ11 splenic B cells transduced with retroviral vectors encoding RNF168^WT or RNF168^R57D were cultured in the presence of LPS/IL-4/RP105 to stimulate CSR. On day 3, cells were irradiated with 2 Gy and fixed 1 h later. Samples were stained for 53BP1 (red) and imaged at 63× magnification. (B) BRCA1^Δ11/Δ11 splenic B cells were transduced with retroviral vectors encoding RNF168^WT, RNF168^R57D or 53BP1^DB and cultured in the presence of LPS/IL-4/RP105 to stimulate CSR. Left panels: overexpression of RNF168 and 53BP1 were confirmed by western blotting. The upper band in the 53BP1 blot corresponds to the endogenous protein. Right panels: two-color flow cytometric analysis of IgG₁ expression in B220-positive cells on day 4; scatter plots for B cells overexpressing RNF168 and its empty vector counterpart were gated on GFP. A representative experiment is shown. (C) Frequency of IgG₁ expression in B220-positive BRCA1^Δ11/Δ11 B cells from three-four independent experiments. (D) Model depicting the differential roles of 53BP1 and RNF168 in physiological and mutagenic NHEJ.