Fig 4.
Ikaros-Cdk9 protein interaction in vitro. (A) Schematic of Ik and Cdk9 protein structures and deletion mutants. Functional domains are designated according to Cdk9 amino acid residues. (B) [35S]methionine-labeled full-length and truncated Cdk9 proteins were resolved on SDS-PAGE gels; arrows indicate predicted molecular weights. (C and D) GST pulldown assay result: [35S]methionine-labeled full-length Cdk9 (C) or truncated Cdk9 (D) proteins were prepared by coupled transcription-translation reactions and tested for interactions with equal amounts of bacterial GST, GST-Ik1, GST-Ik4, or GST-Ik6 (C) or with GST or GST-Ik1 (D) by GST pulldown assay. Bound proteins were analyzed by SDS-PAGE and autoradiography. Input samples represent 20% of binding reaction mixtures. Filled circles, nonspecific bands. (E) Total cell lysates of COS-7 cells transfected with vectors expressing the FH-tagged kinase-dead Cdk9 mutant (FH-Cdk9dn) together with Ik1-FH (Ik1) or Ik1-FH alone were immunoprecipitated with Cdk9 or Ikaros (Ik) antibodies. Co-IP samples were analyzed by Western blotting with Ik or Cdk9 antibodies.