Ebf1 and the Batf/IRF4 complex cooperatively promote the expression of Aicda and Ezh2. (A-B) Ebf1 transduction in B1-8^hi splenic B cells. Cells were transduced with control retroviral vector or retroviral vector expressing Ebf1 on day 1 after differentiation stimuli and sorted on the basis of green fluorescent protein (GFP) expression on day 3. One experiment was performed using 3 mice. (A) RT-PCR analysis of Ebf1 and Ezh2 transcripts. (B) Flow cytometry analysis of intracellular IRF4, 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), IRF4^loIgG1^posi cells (bottom lower), or IRF4^hiIgG1^posi cells (bottom upper). (C-D) Knockdown of Batf and/or Ebf1 in B1-8^hi splenic B cells. Cells were transduced with a control vector or vector targeting Batf (shBatf) and vector targeting Ebf1 (shEbf1) on day 1 after differentiation stimuli. On day 3, cells were sorted on the basis of GFP expression for control, and GFP (shBatf) and/or dsRed (shEbf1) expression for shBatf and/or shEbf1. One experiment was performed using 3 mice. (C) RT-PCR analysis of transcripts of indicated genes. (D) Flow cytometry analysis of surface CD138 and IgG1. For panels A and C, results are presented relative to the abundance of transcripts encoding Β2m, and shown with box-and-whisker plot. *P < .05; **P < .01; ***P < .001. For panels B and D, data are representative of 3 mice, and shown with the means and standard deviation (SD), respectively.