FIG. 1.

EWS is associated with TFIID and copurifies with Pol II. (A) The anti-hTBP (α-TBP) and the anti-hTAF_II100 (α-TAF_II100) MAbs coimmunoprecipitate EWS from a HeLa cell NE. HeLa cell NE was immunoprecipitated (IP) with either an unrelated antibody (lane 1) or a MAb raised against TBP (3G3; lane 2) or hTAF_II100 (2D2; lane 3). Beads were washed and boiled, and bound proteins were analyzed by Western blotting using an antibody raised against the N-terminal domain of EWS that recognizes the endogenous EWS protein in HeLa cell NE (lane 4). M, markers in kilodaltons. (B and C) The anti-EWS antibody coimmunoprecipitates components of the TFIID complex. HeLa cell NE was immunoprecipitated with either the anti-EWS PAb (lanes 2) or the preimmune serum (PI; lanes 1). Beads were washed and boiled, and bound proteins were analyzed by Western blotting using either the anti-EWS antibody (B) or the anti-TBP MAb 3G3 together with the anti-hTAF_II100 MAb 2D2 (C). In panel B, the IgG heavy chain (IgGH) is indicated. (D) EWS copurifies with Pol II. The previously described chromatographic fractions obtained during the purification of Pol II (3) were tested by Western blotting using an antibody raised against either EWS (upper panel) or the fifth-largest subunit of Pol II (hRPB5; lower panel). Hep, Heparin-Ultrogel column; DE, DEAE 5PW HPLC column; φ, Phenyl-5PW HPLC column.