Analysis of class-switched gene expression and germ line transcription. (A) Northern blot analysis of Ig gene transcription. Total RNA was isolated from wt (+/+) and mutant (Δ/Δ) unstimulated (UNS) splenocytes and from LPS- or LPS+IL4–stimulated splenocytes (day 5 after stimulation) and hybridized with a Cμ probe or with a Cγ3 probe that cross-hybridizes with all γ transcripts. The mb1 hybridization was used as a loading control. (B) Analysis of germ line transcription. RT-PCR was performed on wt (+/+) or mutant (Δ/Δ) germ line transcripts from LPS- or LPS+IFNγ–activated splenocyte's RNA (day 4) for Iμ-Cμ, Iγ2b-Cγ2b, or Iμ-Cγ2b or for Iμ-Cμ, Iγ2a-Cγ2a, or Iμ-Cγ2a germ line transcripts, respectively. Single-stranded cDNAs or dilutions thereof (1/5 and 1/25) were subjected to PCR using appropriate primers. PCR products of Iμ-Cγ2b and Iμ-Cγ2a hybrid transcripts were hybridized with the corresponding cDNAs (bottom panels).