BCL-6 is a repressor of IL-6 transcription. (A) RT-PCR analyses of IL-6 mRNA expression in wild-type and BCL-6^-/- macrophages, as well as in BCL-6^-/- macrophages in which either BCL-6 or control GFP was retrovirally expressed. (B) Schematic representation of the 2 IL-6 reporter constructs used in the reporter assay. Locations of the 4 BCL-6 binding sites are also shown. (C) Gel shift analysis using synthetic oligos corresponding to the 4 BCL-6 motifs and nuclear extract from a BCL-6⁺ lymphoma cell line, Ly1. Specificity of the BCL-6/DNA complexes was shown in super-shift assays using a BCL-6 antibody. Sequences of the 4 BCL-6-binding sites studied are given in comparison with the 9-bp consensus BCL-6-binding site. (D) Reporter assays performed in 293T cells with the 2 IL-6 reporters and the indicated amounts of BCL-6 expression plasmids (pmol/plate). The experiments were performed in duplicate, and luciferase activities in all transfections were normalized to that in the absence of BCL-6, which is set as 100 in the graph. (E) Culture supernatants from the reporter assays in panel D were measured for IL-6 secretion by ELISA.