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. 2001 Apr;21(7):2312–2323. doi: 10.1128/MCB.21.7.2312-2323.2001

FIG. 3.

FIG. 3

Purification of dMediator. (A) Outline of dMediator purification. The numbers indicate the concentrations (millimolar) of potassium acetate (heparin-Sepharose, SP-Sepharose, and Mono S) or potassium phosphate (hydroxyapatite). (B) Immunoblot and transcriptional (Trxn) analyses of Mono S fractions. Equal amounts of the input (I) samples were loaded on the Mono S column, and the eluted fractions (numbers above the lanes) were resolved by SDS-PAGE and immunoblotted with the Abs indicated at the left. Mono S fractions 18 to 32 were added to the transcription reactions containing the Gal4-VP16 protein and soluble nuclear fraction immunodepleted with anti-dSOH1. (C) Transcription assay of dMediator activity. The Gal4-VP16 protein and soluble nuclear fraction (SNF) immunodepleted with anti-β-galactosidase (SNFmock) or anti-dSOH1 (SNFαdSOH1) were used in transcription reactions as in Fig. 2C. The Mono S peak fraction (#24) and immunopurified dMediator (IP pellet) were added to the reactions as described above.

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