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. 2001 Jan;21(1):1–15. doi: 10.1128/MCB.21.1.1-15.2001

FIG. 6.

FIG. 6

Binding assays using C. elegans TFIIEα. (A) Binding of hTFIIEα to the various general transcription factors. All of the human general transcription factors (400 ng each) except for hTFIIEα, the TBP activation factors of TFIID, and the TFIIH subunits were fused to GST and expressed in bacteria. GST-pull down assays were carried out as described for Fig. 5A using 200 ng of 6H-hTFIIEα. After Western blotting, bound hTFIIEα was detected with anti-hTFIIEα antibody. Lane 1, GST alone (G); lane 2, GST-TFIIB (B); lane 3, GST-hTFIIEβ (Eβ); lane 4, GST-TFIIFα (Fα); lane 5, GST-TFIIFβ (Fβ); lane 6, GST-TBP (T); lane 7, GST-TFIIAα (Aα); lane 8, GST-TFIIAβ (Aβ); lane 9, GST-TFIIAγ (Aγ). Arrows indicate the position of 6H-hTFIIEα (hIIEα). (B) Binding of ceTFIIEα to the various general transcription factors. GST-pull down assays were carried out as described for panel A using 200 ng of HA-ceTFIIEα. After Western blotting, bound HA-ceTFIIEα was detected with anti-HA monoclonal antibody (12CA5). An arrow indicates the position of HA-ceTFIIEα (ceIIEα). (C) Binding of hTFIIEα to the TFIIH subunits. Assays were carried out as described for panel A. Four hundred nanograms of each GST-fused TFIIH subunit, with GST alone (lane 1) as a control, were used to examine binding to hTFIIEα. An arrow indicates the position of 6H-hTFIIEα (hIIEα). (D) Binding of ceTFIIEα to the TFIIH subunits. Assays were carried out as described for panel C, except that HA-ceTFIIEα was used instead of 6H-hTFIIEα. An arrow indicates the position of HA-ceTFIIEα (ceIIEα). Molecular mass markers are indicated to the left.