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. 1998 Sep;18(9):5355–5363. doi: 10.1128/mcb.18.9.5355

FIG. 3.

FIG. 3

(A) Western blot analysis of the fractions derived from phosphocellulose and DEAE-cellulose columns. Western blots of eluates of 0.1 (0.18 mg of protein per ml), 0.3 (0.12 mg of protein per ml), 0.5 (0.08 mg of protein per ml), and 1.0 M KCl (0.16 mg of protein per ml) from a phosphocellulose column (left panel) and 0.1 (0.03 mg of protein per ml) and 0.35 M KCl (0.18 mg of protein per ml) eluates of the DEAE-cellulose column (right panel) derived from the phosphocellulose 0.5 M KCl fraction are shown (see Materials and Methods). Each eluate (20 μl) was loaded on an SDS–5 to 20% polyacrylamide gel for Western blot analysis. Each blot was analyzed for CDK8, cyclin C (cyc C), hSrb7, BAF190, BAF47, the largest subunit of RNAPII (RPB1), the 89-kDa subunit of TFIIH (ERCC3) (TFIIH p89), the large subunit of TFIIF (RAP74), the small subunit of TFIIE (p34), TFIIB, and the histone acetyltransferases CBP and PCAF. (B) Affinity-purified CDK8 complex contains hSrb proteins, Swi-Snf subunits (BAFs), and substoichiometric amounts of RNAPII but not GTFs, the corepressor DRAP1, or the coactivator PC4. Western blots of input (I), flowthrough (FT), and eluate (Elute) of anti-CDK8 (CDK8 col) and control antibody (con. col) columns are shown. Blots were probed for BAF190, BAF170, BAF155, BAF60, BAF47, CDK8, cyclin C, hSrb7, RPB1, TFIIF (RAP74), TFIIH (p62), TFIIE (p56), TBP, TFIIB, DRAP1, and PC4. (C) A silver-stained SDS-polyacrylamide gel containing the affinity-purified CDK8 complex and the Swi-Snf complex shows multiple common polypeptides. A silver-stained gel containing affinity-purified CDK8 complex (left) and Swi-Snf complex (right) was aligned to show common polypeptides (>29 kDa). Another silver-stained gel containing bands from 29 to 10 kDa showing polypeptides derived from the affinity-purified CDK8 complex is at the bottom of the left lane. Polypeptides were identified by Western blot analysis as indicated. Other polypeptides in this fraction which are common to the polypeptides present in the DEAE-cellulose flowthrough fraction-derived affinity-purified CDK8 complex were identified by microsequencing (67a).