Fig 5.

Nonfunctional VDJ- and DJ-rearranged IgH mRNAs are efficiently degraded by NMD. mRNA levels from nonproductively rearranged IgH^frVκ or IgH^wt alleles in LPS-stimulated B cells treated or not with CHX on day 4 were determined. (A) Northern blotting was performed by using the frVκ probe that is specific for Vκ6 segments and allows the simultaneous identification of IgH^frVκ and Igκ (normalization) mRNAs. The stimulation of B cells with LPS in the absence of additional cytokines induces preferential class switch recombination (CSR) to γ2b and γ3 isotypes. (B) Nonproductive mRNAs from switched IgH^frVκ alleles were assessed by semiquantitative PCR using primers frVκ-for and Cγ3-rev (primer set k/h in Fig. 1A). (C) IgM^a-positive B cells were sorted from 129/B6 F1 (IgH^a/b) mice (as described in the legend to Fig. 3D) and stimulated with LPS for 4 days. Nonproductive VDJ-rearranged mRNAs from switched IgH^b alleles were then analyzed by using a consensus VH₇₁₈₃ forward primer (VH₇₁₈₃-for) and allotype-specific Cγ2b reverse primers (primer set a/i in Fig. 1A). (D) Degradation of DJ-Cμ mRNAs by NMD was assayed with wt mice by semiquantitative RT-PCR using a consensus forward primer (DHL-for) located at the 5′ end of most DH segments (primer set d/g in Fig. 1A). Representative results from 3 to 4 different mice (A, B, and D) or from 2 independent cell sorting experiments (C) are shown.