Figure 4.

Uracil residues in the S_μ region after ex vivo stimulation of mouse splenic B cells with LPS and IL-4. (a) Map of the S_μ region. Dotted oval indicates the scope of SHM; S_μ primers amplify 858 bp by quantitative PCR; gray bar indicates position of the probe to hybridize a 2.6-kb fragment generated by digestion with HindIII (H). (b) Immunoblot analysis (top) of AID expression in Ung^−/− mouse splenic B cells treated with LPS and IL-4. Below, quantification of band intensity, presented relative to that of β-actin. Data are representative of three experiments (error bars, s.e.m.). (c) Quantitative PCR analysis of the amplification of S_μ in intact DNA from Ung^−/− cells (presented as in Fig. 3b). *P = 0.02 (two-tailed t-test). Data are representative of four experiments (error bars, s.e.m.). (d,e) Southern blot analysis (top) of intact DNA isolated from Ung^−/− cells (d) or Ung^−/−Aicda^−/− cells (e), digested with HindIII, treated with APE1 with or without UNG, separated by electrophoresis through in alkaline agarose gels and blotted with probes to S_μ and Dhfr. Below, quantification of uracil in treated samples, standardized to Dhfr results and presented relative to that of untreated samples. *P = 0.02 (two-tailed t-test). Data are from three independent experiments (error bars, s.e.m.).