FIG. 3.

Immunodepletion of Mediator from HNEs. HNEs were immunodepleted with a mixture of antibodies to Med220 and Med33 (depleted) or mock depleted with control antibody (control). Aliquots of the original extract (input), Mediator-depleted extract, or control-depleted extract were analyzed by immunoblotting with an array of antibodies to Mediator subunits (Med220, Med150, Med130, Med78, Med34, Med33, and Cdk8) (A, left panel), components of the RNA pol II transcriptional machinery (the large subunit of RNA pol II, TBP, TFIIB, TFIIE p34, and TFIIF p74) (B), and coactivators and chromatin remodelers (p300, CBP, PCAF, SRC1, SRC2, SRC3, Brg1, and Brm) (C). The immunoprecipitated or coimmunoprecipitated material (IP or Co-IP, respectively) was also analyzed by immunoblotting for the presence of Mediator subunits (A, right), as well as RNA pol II, p300, CBP, and TBP (D). Note that the Mediator subunits are designated according to the unified nomenclature proposed by Rachez and Freedman (41).