Fig. 6. TR and v-ErbA recruit HDAC3. (A) TR enriches for N-CoR and HDAC3, but not RPD3. A large aliquot of Xenopus egg extract was adjusted to 50 mM NaCl and 0.1% NP-40 and incubated with a preparation of bead-immobilized GST–TR (lanes 3 and 4) or GST (lane 2). Where indicated, 2.5 µM T₃ was included in the reaction. Following washing as described in Figure 4D, proteins associated with the beads were analyzed by immunoblotting against N-CoR (top panel), HDAC3 (middle panel) and RPD3 (bottom panel). (B) v-ErbA-dependent enrichment for N-CoR in the immunoprecipitate is accompanied by an enrichment for HDAC3. One half of the immunoprecipitate described in the legend to Figure 5A was resolved on a 4–12% Laemmli SDS–PAGE, and analyzed by autoradiography (top panel) and western blotting with antibody against N-CoR (middle panel) and human HDAC3 (bottom panel). As in Figure 4B, the longer (10 min) exposure used in the bottom panel reveals both the ³⁵S-labeled antigen directly targeted in the immunoprecipitation as well as chemiluminescence-derived signal from the antibody used in the western blot. Low efficiency of the anti-HDAC3 antibody prevents the visualization of HDAC3 in the 0.5 oocyte equivalents of extract used in the input lanes (lanes 1 and 3). (C) N-CoR and HDAC3 co-immunoprecipitate. A preparation of bead-immobilized rabbit antiserum against N-CoR (lanes 4 and 7), the cognate pre-immunization serum (lanes 3 and 6) or the beads with no antibody (lanes 2 and 5) was incubated in Xenopus egg extract adjusted to 50 mM NaCl and 0.1% NP-40. The proteins associating with the antibody were eluted with glycine and analyzed for the presence of HDAC3 by immunoblotting with rabbit-derived anti-HDAC3 serum and extended duration ECL reagents, followed by visualization and quantitation on a CCD camera-equipped ChemiImager-4400 low light imaging system. The small amount of rabbit anti-N-CoR antibody released from the beads (lanes 2–4) yields Ig heavy chain signal at 50 kDa (lanes 2–4), i.e. at the molecular weight of HDAC3; quantitation indicates a 60-fold greater signal in lane 7 (anti–N-CoR antibody + egg extract) than in lane 4 (anti–N-CoR antibody only). Lane 1 contains an aliquot of egg extract analyzed on the same gel and probed with conventional ECL reagents, followed by autoradiography.