Genomic analyses of Fra-2 targets in B cells. (a) ChIP-seq analysis of the binding of Fra-2 at Ebf1 promoter (n = 3) performed in B220+ B cells from control mice. (b) Real-time PCR analyses of mir125b, lyn37, and pdk3 in pro–B cell cultures of Fra-2ΔB cell and littermate control cells stimulated with IL-7 for 5 d (n = 4). (c) PCR loading fragments of ChIP analyses from littermate control cells for Irf4 promoter after immunoprecipitation of the chromatin with AP-1 protein antibodies. Arrows indicate primers amplifying fragments for the TRE elements. Real-time PCR analyses of ChIP in Fra-2ΔB cell and littermate control B220+ B cells for Irf4 promoter with AP-1 antibodies (n = 3). (d) PCR loading fragments of ChIP analysis for Ikaros promoter after Fra-2 and Irf4 ChIP and real-time PCR quantification of the binding potential of AP-1 members in Fra-2ΔB cell and littermate control cells (n = 3). (e) Coimmunoprecipitation of IRF4 and Fra-2 proteins in B220+ bone marrow B cells (n = 3). (f) Western blot analysis of Irf4, Foxo1, and actin proteins in Fra-2ΔB cell and littermate control B cells (n = 3). (g and h) PCR loading fragments of ChIP analysis of Foxo1 promoter after AP-1 antibody ChIP (g) and real-time PCR quantification (h). (i and j) Real-time PCR quantification of ChIP analysis for IL7Rα (i) and (j) promoters after ChIP with Foxo1 antibody in Fra-2ΔB cell and littermate control B cells. (k) Schematic representation of genes transcriptionally regulated by Fra-2. pBCR, pre-BCR. Bars represent mean values ± SD (n = 3). Data show one representative out of three independent experiments. Statistical analysis was performed using Student’s t test; *, P < 0.05. Beads-Fra-2, beads treated with Fra-2 antibody; Beads-Iso, beads treated with control isotype antibody; c-Iso, control isotype antibody; Inp, input; IP, immunoprecipitation with Fra-2 antibody.
