Cofactors that interact with the retinoid receptors in APL cells.
(A) Schematic diagram of the double cistronic expression plasmid. The RXR LBD is fused with GST, and the RAR LBD is tagged with 6 histidine residues. Both cDNAs are driven by a Tac promoter under the control of the lac operator. The ribosome-binding site (rbs) preceding each cistron allows the coexpression of both proteins. Proteins can be purified by either glutathione Sepharose or Ni2+ NTA agarose. (B) Ligand-inducible interactions between GST-RXR LBD/RAR LBD and a group of proteins isolated from APL cell line NB4 nuclear extracts. Immobilized GST-RXR LBD/RAR LBD was incubated with 1.2 mg NB4 nuclear extract in the absence (−) or presence (+) of 5 μmol/L all-trans retinoic acid (t-RA) (lanes 2, 3). GST alone was used as a control (lane 1). Bound proteins were eluted, separated by SDS-PAGE, and visualized by nitrate silver staining. Approximate molecular weights of the interacting proteins are indicated at right. (C) Binding of the complex to retinoid receptors in response to selective ligands. Protein interactions were examined in the absence (−) or presence of 1 μmol/L t-RA (lane 2), 1 μmol/L RXR-specific ligand LG100153 (lane 3), and 1 μmol/L RAR-specific ligand LG100272 (lane 4).
