Figure 5. Impaired class switch recombination in Rnf168^−/− mice.

(A) Serum immunoglobulin levels in 6–8-week-old and 9–12-month-old Rnf168^−/− mice. The data are presented as the mean ± SEM (n = 3–5). (B and C) FACS analysis of IgG1 (B) or IgG3 (C) and IgM expression on B220⁺ B-cells (left panels) and average percentages of IgG1⁺ or IgG3⁺ B-cells (right panels) from Peyer's patches. Three independent experiments were performed. (D) Secreted immunoglobulins were analyzed in the supernatants of B-cell cultures after 4 days of in vitro stimulation with LPS with or without IL-4. The data are presented as the mean ± SEM from three independent experiments. (E) FACS analysis of IgG1 expression on CFSE labeled B-cells stimulated with anti-CD40 plus IL-4 for 4 days (left panels) and average percentages of IgG1 switched cells (right panel). Five independent experiments were performed. (F) CFSE staining profiles of B-cells stimulated with anti-CD40 antibody plus IL-4 for 4 days (upper panel) and percentages of IgG1⁺ cells that had undergone the indicated number of cell divisions (lower panel). Representative data are shown from three independent experiments. (G) Schematic representations of WT and mutants Rnf168 cloned into the MSCV-IRES-GFP vector. (H) B-cells infected with the indicated ecotropic retroviruses [MSCV-mutated or full-length (FL) Rnf168-IRES-GFP] were stimulated with LPS plus IL-4 for 4 days, and the level of CSR to IgG1 was examined by FACS. Data are presented as the mean ± SEM of four independent experiments. * indicates p<0.05 compared to WT. (I) Representative DC-PCR for Sμ-Sγ1 recombination is shown from three independent experiments. nAchR served to normalize for the amount of input DNA. Fivefold serial dilutions were used as templates. H₂O: no input DNA.