Reconstituting the expression of either Ikaros or Aiolos is sufficient to down-regulate pre-BCR in IRF4,8^−/− pre-B cells. (A) IRF4,8^−/− pre-B cells were infected with virus expressing control, IRF4, Ikaros, and Aiolos. At 48 hours after infection, the cells were stained with antibodies against pre-BCR and CD19 and analyzed by FACS. The GFP^hi cells were gated and further analyzed. The numbers represent percentages of the gated cells. (B) Relative expression levels of Ikaros and Aiolos were assessed by real-time PCR. The GFP^hi and GFP^lo-int cells were isolated via sorting from control-vector, Ikaros-, and Aiolos-infected cells (48 hours after infection). In addition, GFP^hi cells from IRF4ER-infected cells were isolated via sorting 24 hours after tamoxifen treatment. Ikaros-infected cells were used to measure Ikaros expression, whereas Aiolos-infected cells were used solely to measure Aiolos expression. However, the expression levels of both endogenous Ikaros and Aiolos were measured in the control-vector and IRF4ER-infected cells (GFP^hi). The value in the control-vector infected cells was set as 1. (C) Western blot analysis of Ikaros protein from GFP^hi and GFP^lo-int cells isolated from Ikaros-infected cells, and the sorted control-vector and IRF4ER-infected cells (GFP^hi). (D) The infected cells (GFP^hi) were sorted at different time points, and total RNA was isolated for real-time PCR analysis. λ5 expression was measured, and the value of control-vector infected cells at 0 hours was arbitrarily set as 1. The values are average and SD of 3 independent experiments.