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. 2004 Feb 12;23(4):719–727. doi: 10.1038/sj.emboj.7600111

Figure 5.

Figure 5

Analysis of a recombinant complex containing seven TAFs. (A) Seven TAFs (hTAF4, 5, 6, 9, 10, 12 and mTAF8) were coexpressed in Sf9 cells and the TAF10-containing complexes were purified using a TAF10-specific antibody. The bound complexes were eluted and separated by gel filtration on a Superdex 200 column. (A) SDS–PAGE analysis and silver staining of the collected fractions showing the formation of a homogeneous complex in fraction 14 with an estimated molecular weight of 800 kDa. The elution of molecular weight standards is indicated (top). (B) Western blot analysis showing that the seven TAFs coelute in the peak fractions of the Superdex 200 gel filtration profile. (C) Western blot analysis of anti-TAF10 immunoprecipitation (IP) experiments. Lane 1, coexpression of the seven TAFs; lane 2, TAF5 is missing in the coexpression; lane 3, TAF10 is missing in the coexpression. (D) Electron microscopic observation of the peak fraction 14 reveals a homogeneous population of complexes in negative stain. (E) Image analysis of the seven-TAF complex reveals a TFIID-like trilobed structure. The bar represents 50 nm in (D) and 13.5 nm in (E).