Knockdown of Ikaros but not Aiolos resulted in defect of plasma cell differentiation. (A-B) Knockdown of Ikaros or Aiolos in B1-8^hi splenic B cells. Cells were transduced with a control vector or vector targeting Ikaros (shIkzf1) or Aiolos (shIkzf3) on day 1 after differentiation stimuli, then sorted on the basis of GFP expression on day 3. One experiment using 3 mice. (A) RT-PCR analysis of Ikzf1 and Ikzf3 transcripts. Results are presented relative to the abundance of transcripts encoding Β2m, and shown with box-and-whisker plot. (B) Flow cytometry analysis of intracellular IRF4 and CD138 and IgG1. Numbers adjacent to outlined areas indicate percent IRF4^loCD138^nega cells (top lower), IRF4^hiCD138^nega cells (top upper left), IRF4^hiCD138^posi cells (top upper right), or IgG1^posi cells (bottom). Data are representative of 3 mice, and shown with the mean and SD, respectively. (C) The binding of IRF4 or Ikaros to their regulatory regions at the Ebf1, Haao, and Setd2 loci. B1-8^hi splenic B cells were stimulated for 48 hours, and ChIP assay was performed using control IgG, α-IRF4, or α-Ikaros, followed by quantitative PCR analysis. Binding enrichments is presented relative to input DNA. The average enrichment and SD is from 3 independent experiments for α-IRF4, and 2 independent experiments with technical duplicate for α-Ikaros. For panels A and C, *P < .05; **P < .01; ***P < .001.