Figure 1.

Purification, identification, and verification of SMRT complex components. (a) Pooled SMRT carboxy-terminal monoclonal antibodies are not cross-reactive with N-CoR. In vitro-translated SMRT, N-CoR, or unprogrammed rabbit reticulocyte lysate (RRL) control were subjected to immunoblot analysis with five pooled SMRT monoclonal antibodies or an anti-N-CoR monoclonal antibody. (b) Strategy to obtain SMRT-associated polypeptides. (c) SDS-PAGE and silver-staining analysis of SMRT complex purified as in b from HeLa nuclear extract. SMRT complex components are indicated by arrows. Asterisk denotes a band that was not reproducibly observed in eluates from the SMRT column. (d) Identity of SMRT complex subunits. Peptide sequences obtained by microsequencing are underlined. (e) Immunoblot analysis of SMRT complexes purified independently. Components isolated from nuclear extract or as in b were verified by immunoblot with anti-SMRT, mouse anti-TBL1, anti-HDAC3, anti-Sin3A, and anti-HDAC1. (f) HDAC assay of SMRT purified as in b.