Fig 1.

Sequence of mammalian MED8 and homology to Sch. pombe and S. cerevisiae MED8 proteins. (A) Amino acid sequence of human mMED8. The Elongin BC-box is boxed. Peptide sequences derived from ARCp32 by Tjian and coworkers (2) are underlined. (B) Alignment of the N termini of human, Sch. pombe, and S. cerevisiae MED8 proteins, generated by using the ALIGNX program from Vector NTI. (C) Anti-FLAG immunoprecipitations were performed essentially as described (31) with nuclear extracts prepared according to Dignam et al. (36) from ≈2.5 × 10⁹ HeLa cells (lane 1) or HeLa M10 cells (25) stably expressing FLAG-tagged NUT2 (lane 2). Nuclear extracts were adjusted to 300 mM KCl and 0.5% Triton X-100 and incubated with M2-agarose beads (Sigma). Beads were washed with buffer containing 50 mM Hepes⋅NaOH (pH 7.9), 5 mM MgCl₂, 20% glycerol, 300 mM KCl, and 0.1% Triton X-100, then eluted with the same buffer containing 100 mM KCl, 0.05% Triton X-100, and 0.2 mg FLAG peptide (Sigma) per milliliter. Precipitated proteins were analyzed by blotting with anti-FLAG, anti-mMed8, or anti-Trap80 antibodies. The positions and relative molecular masses in kDa of protein size markers are indicated on the left (Prestained Precision Protein Standards, Bio-Rad).