Fig. 6

NFKB1 (NF-kappa B p50) interacts with Ikaros and this interaction is essential for recruiting Rel-NF-kappa B family TFs to M4 motif harboring DNA. a Immunoblot analysis of DNA affinity chromatography experiments using two DNA ligands: mono-M4 WT and mono-M4 MT. Molt4 control or Molt4-Ikaros KD nuclear extracts served as input. In the absence of Ikaros, protein levels of NFKB1 (NF-kappa B p50) were virtually not affected (lane 4 vs 1) but its binding to the M4 column was completely abrogated (lane 5). In contrast, the binding of the two other established M4-binders (THAP11 and HCF1) were robustly maintained (lane 2 and 6). b Anti-NF-kappa B p50 (NFKB1) IP (immunoprecipitation) co-precipitated Ikaros in the presence (lane 4,5) and absence (lane 2, 3) of the highly efficient nuclease bezonase. c Cross-linked streptactin pulldown experiment demonstrates co-purification of StrepII-tagged Ikaros (see the text) with NFKB1 (NF-kappa B p50) protein (lane 4), whereas the nuclear protein kinase CDK9 that was reported to interact with NFKB1 (NF-kappa B p50) (see text) was not present in the cross-linked complex (compare lane 1, 2 with 3, 4). d Benzonase nuclease digestion control assay: the control 40 bp duplex DNA or control salmon sperm DNA were mixed with Molt4 nuclear extracts in the presence or absence of benzonase at 4 °C (5 h to overnight). The whole samples were analyzed by an 8 % (19:1) native TBE-PAGE gel that allows separation of DNA down to the size of 10 bps