Zfp521 antagonizes Ebf1 activity. (A) Ebf1 was immunoprecipitated with α-Ebf1 antibody. Mouse IgG was used as control. The immune complexes (left) and 10% input were blotted with α-Ebf1 and α-Zfp521 as indicated. (B) HA-Zfp521 and Flag-Ebf1 proteins were overexpressed in 293T cells. Proteins were immunoprecipitated with α-HA, and the immune complexes (two top panels) and 5% of the original cell lysates (two bottom panels) were blotted with α-HA and α-Flag as indicated. (C) Ebf1-responsive B29-luc plasmid was transfected into 293T cells together with Ebf1, Zfp521, or both. Luciferase activity was normalized to cotransfected Renilla activity. (D) The expression of Ebf1 target genes Cxcl12, Ccl9, and Pparγ was measured by qRT-PCR in Zfp521^−/−, Zfp521_hOC^−/−, and respective control calvarial cells cultured for 7 d in OM. (E) Schematic presentation of Zfp521 domain structure and mutants. (F) HA-Zfp521 and HA-Zfp521ΔN were overexpressed in 293T cells and immunoprecipitated with anti-HA antibody. The Western blot was performed with antibodies against endogenous NuRD complex proteins (MTA1, MTA2, and HDAC2) and anti-HA antibody. (G) B29-luc plasmid was transfected into 293T cells together with Ebf1 and Zfp521 or Zfp521-deletion mutants as indicated. Luciferase activity was normalized to cotransfected Renilla activity. (H) B29-luc plasmid was transfected into 293T cells together with Ebf1 and Zfp521 or Zfp521ΔC as indicated. Luciferase activity was normalized to cotransfected Renilla activity. (I) Ccl9-luc plasmid was transfected into 293T cells together with Ebf1 and Zfp521 or Zfp521-deletion mutants as indicated. Luciferase activity was normalized to cotransfected Renilla activity. All data are means ± SD. Similar results were obtained in at least three independent experiments, and the assays were performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.