Figure 9

RXR binds the amino terminus of hNPDC-1. Approximately 10 μg of recombinant S-tagged hNPDC-1 or NPDC-1 deletion mutant (A) was pre-coupled to S-protein to generate a 50% slurry of NPDC-1-beads. Reactions containing 30 μl NPDC-beads, 5 μg of recombinant GST-RXRα and 300 μl of 1X gel-shift buffer were incubated at 4°C for 1 hour with rotation. S-protein pellets were collected by centrifugation and washed 3 times in gel-shift buffer. Proteins were released from the pellet by resuspending S-protein agarose in 2X SDS-PAGE sample buffer. The amount of GST-RXRα bound to beads was analyzed by Western blotting onto nitrocellulose filters and probing blots with anti-RXRα (B, top panel). To normalize for unequal expression of the various deletion mutants, duplicate reactions without GST-RXRα were performed and subjected to coomassie staining instead of immunoblotting, and NIH Image Software was used to normalize the bands in the immunoblot (B, top panel) to the amount of S-tagged NPDC-1 precipitated in the coomassie stained gel. Normalized binding is depicted in B, bottom panel.