Figure 1. IKAROS interacts with the newly characterized NuRD-P-TEFb complex.

A) Purification of Flag-HA-Ik associated proteins. Nuclear extracts from Jurkat cells carrying the pOZ-N-Flag-HA-IKAROS-IRES-IL2R vector and expressing a double tagged IKAROS (Flag-HA-Ik) or Jurkat cells expressing the pOZ-N-Flag-HA-IRES-IL2R empty vector (Mock) were used for sequential immunoaffinity purification using Flag- followed by HA-conjugated matrix. A fraction of the purified complexes were loaded on SDS-PAGE and silver stained; E1: first HA elution; E2: second HA elution; MW: molecular weights (in KDa); the Flag-HA-Ik protein is indicated by an arrow; B, C) Molecular weight fractionation of the IKAROS-associated complexes. Flag-HA-Ik immunoaffinity purified complexes (panel B) or nuclear extracts (panel C) were fractionated using a Superose 6 10/300GL column; 0.5 ml fractions were collected, TCA-precipitated and loaded on SDS-PAGE for western blot analysis; immunoblots were probed with the antibodies indicated at the bottom or on the right side of the panels; the 37 KDa APE1 nuclear protein was used as control; D) Protein co-immunoprecipitation of Jurkat cell nuclear extracts. Immunoprecipitations were carried out with the antibodies indicated on top of each panel; these antibodies were specific for: IKAROS, the general P-TEFb activator BRD4, NuRD-associated proteins (Mi2, RBBP4, MTA2, MBD3) or P-TEFb components (CDK9, CYCT1). Immunoblots were probed with the antibodies indicated at the bottom of the panels. Input samples represent 2% of nuclear extracts; IgG: isotype-matched immunoglobulins; asterisk: IgG light chains; filled dots: non-specific bands; n≥3; E) The nuclear protein PCNA was used as negative control for the immunoprecipitation procedure; F) Summary of the most relevant protein-protein interactions; +: strong interaction; +/−: weak interaction; −: no interaction; nd: not determined.