Class switch recombination activity in B-cell specific <i>Ssb1</i>-deleted mice.

<p><i>Ssb1 ^fl/fl</i> mice were crossed with a <i>Cd19</i>-Cre transgene expressing C57BL/6J mice to specifically delete <i>Ssb1</i> in B cells. Splenic B cells were isolated and stimulated for 2, 3, and 4 days using anti-Cd40 antibodies plus IL-4 to induce CSR to IgG1. (A) Western blotting of whole cell extracts showed loss of Ssb1 protein in stimulated B cells from <i>Ssb1</i> knockout (<i>Ssb1</i> KO; <i>Cd19</i>Cre⁺: <i>Ssb1^−/−</i>) mice. Equal amounts of stimulated B cell extracts from heterozygous <i>Ssb1</i> (<i>Ssb1</i> Het; <i>Cd19</i>Cre⁺: <i>Ssb1^+/−</i>) and wild-type <i>Ssb1</i> (<i>Ssb1</i> WT; <i>Cd19</i>Cre⁺: <i>Ssb1^+/+</i>) mice were included for comparison. Equal loading was confirmed by probing for Hsp90α. (B) Western blotting of Ssb2 levels in B cells from <i>Ssb1</i> WT and <i>Ssb1</i> KO mice. (C) FACS analysis of CSR to IgG1 over time in stimulated B cells from mice of the indicated genotypes. Stimulated splenic B cells from <i>AID^−/−</i> (<i>AID</i> KO) mice served as a negative control. (D) Summary statistics of CSR activity to IgG1 and viability on day 3 of stimulation. Mean and S.E.M. from three independent experiments are shown. No statistically significant differences (two-tailed unpaired <i>t</i>-test) were found. (E) Switch region junction analysis. Sm-Sg1 junctions were amplified from IL4 plus anti-CD40 stimulated primary B cells (day 4) and sequenced. Percentage and total numbers of direct or microhomology-mediated joints are indicated. nt, nucleotides.