Supplementary Figure 7: Enhanced binding of IRF5 to ABC regulatory regions in the absence of the SWEF proteins

(a) ChIP assays were performed with either an IRF5 antibody or a T-bet antibody on CD23⁺ B cells purified from WT, DKO, and Cd21-Cre Irf5^fl/– DKO mice and stimulated with αIgM (5 μg/ml) and αCD40 (5 μg/ml) (top panel) or with αIgM (5 μg/ml), αCD40 (5 μg/ml) and IL-21 (50 ng/ml) (bottom panel) for 2 days. qPCR for the ABC specific ATAC-seq peak at the Il6 TSS or the Zeb2 Exon 8 locus was performed. Data are representative of 2 independent experiments. Mean ± SEM is shown (n = 2 mice/grup). ns: not significant; ** P = 0.0079. (One-way ANOVA followed by Bonferroni’s multiple comparisons test). (b) Nuclear extracts were prepared from CD23⁺ B cells purified from WT, DKO and Cd21-Cre Irf5^fl/– DKO female mice (8–10 weeks of age) stimulated with αIgM (5 μg/ml), αCD40 (5 μg/ml) +/− IL-21 (50 ng/ml) or imiquimod (1 μg/ml) for 3 days. Extracts were analyzed by immunoblotting with pSTAT3, STAT3, IRF5, and HDAC1 antibodies. Data are representative of 2 independent experiments. (c) Quantification of immunoblot in (b) by densitometry. P-STAT3, STAT3 and IRF5 were normalized to HDAC1 density. Mean and individual values of 2 independent experiments is shown. (d) Nuclear extracts were prepared from cells stimulated as in a. for 2 days and subjected to ONP assay with a biotinylated oligonucleotide from the Il6 TSS. Precipitated proteins were analyzed by immunoblotting with IRF5 and T-bet antibodies. Data are representative of 2 independent experiments. (e) Binding of IRF5 to the ONP shown in Fig. 7d. 293T cells were transiently transfected with various IRF5 and T-bet constructs as indicated. Nuclear extracts were prepared and subjected to ONP assay with a biotinylated oligonucleotide from the Cxcl10 cluster. Precipitated proteins were analyzed by immunblotting with a FLAG antibody. Data are representative of 2 independent experiments. (f) and (g) 293T cells were transiently transfected with various DEF6, SWAP-70, and IRF5 constructs as indicated. Immunoprecipitations were performed using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting using an anti-IRF5 or anti-HA antibody. Data are representative of 2 independent experiments with similar results. (h), (i), and (j) 293T cells were transiently transfected with various constructs as indicated. Immunoprecipitations were performed using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting using an anti-FLAG, T-bet, DEF6 or HA antibodies. Data are representative of 2 independent experiments with similar results.