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. 2009 Dec 21;206(13):3031–3046. doi: 10.1084/jem.20091892

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© 2009 Ueda et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — XPD mutations disrupt TFIIH integrity. (A) Production in SF9 insect cells of the recombinant mutated forms of XPD tagged with either the His-tag (His-XPD/WT, /R683W, Q452X, and 199insPP) or the Flag-tag (Flag-XPD/WT and /I455del). Whole-cell extracts were resolved by SDS-PAGE and blotted with antibodies raised against either the His- or the Flag-tag. Arrows indicate the theoretical molecular weight of each XPD mutated form. The results are representative of four independent experiments. (B) Purification of the recombinant TFIIH (rIIH). Insect cells were infected with baculoviruses overexpressing the subunits of TFIIH including either WT or mutated XPD, and complexes were immunoprecipitated by using antibody (Ab) directed toward the p44 subunit of the core TFIIH in low salt conditions (50mM KCl). After elution with a synthetic peptide recognized by Ab-p44, equal amounts of purified rIIHs were then resolved by SDS-PAGE and blotted with antibodies against XPB, p62, p52, cdk7 and cyclin H subunits of TFIIH. His-XPD/WT, /R683W, /Q452X, /199insPP, and Flag-XPD/WT, /I455del were visualized with antibodies raised against either the His- or the Flag-tag, respectively. Arrows indicate the different forms of XPD. The results are representative of five independent experiments. (C) Immunoprecipitation with Ab-p44 of the various rIIH in a higher salt condition (150 mM KCl). rIIHs immunoprecipitated with Ab-p44 cross-linked on agarose beads were boiled, resolved by SDS-PAGE, and blotted with antibodies against p62, cdk7, cyclin H, and either the His- or the Flag-tag. Arrows show the different forms of XPD. HC., heavy chain of Ab-p44; LC, light chain of Ab-p44; * indicates a nonspecific band. The results are representative of three independent experiments. (D) Infected Sf9 cell lysates containing either WT or mutated XPD as indicated at the top of the panel, were incubated with (+ p44) or without (− p44) WT p44, immunoprecipitated to agarose beads and further washed with 350 mM KCl. The immunoprecipitated fractions were then resolved on SDS-PAGE, followed by immunoblotting using Ab-p44 and either anti-His or anti-Flag antibodies. * indicates a nonspecific band. The results are representative of three independent experiments.