IRF4,8 suppress SLC expression and down-regulate pre-BCR in pre-B cells. IRF4,8^−/− pre-B cells were isolated from the bone marrow of IRF4,8^−/− mice and were cultivated in Opti-MEM medium containing 5 ng/mL IL-7. The cells were infected with virus expressing ER, IRF4ER, or IRF8ER. After 24 hours, tamoxifen (1 μm) was added, and the cells were analyzed at different time points as indicated. (A) The cells were analyzed by FACS 48 hours after tamoxifen addition. The expression of pre-BCR was analyzed in cells expressing a high level of GPF (GFP^hi). The numbers in the quadrants represent percentages of gated cells. (B) Time-course study was conducted after tamoxifen treatment to analyze λ5 expression in ER- and IRF4ER-infected cells. The λ5 expression in gated (GFP^hi) cells was analyzed by FACS. The numbers in the quadrants represent percentages of gated cells. (C,D) Real-time PCR analysis on expression of λ5 and Vpre-B mRNA. The infected cells (GFP^hi) were isolated via sorting at different time points after tamoxifen treatment, and total RNA was isolated for PCR analysis. The value of ER-infected cells at 0 hours was arbitrarily set as 1. Each primer set was independently repeated 3 times, and average values and standard deviations were calculated. (E) IRF4ER- and IRF8ER-infected cells (GFP^hi) were isolated by sorting for protein extraction. Western blot analysis was performed to measure the expression of IRF4ER and IRF8ER in the sorted cells and IRF4 and IRF8 in the cultivated wild-type pre-B cells.