FIG. 5.

S. cerevisiae contains two Kin28-containing complexes. S. cerevisiae strains containing His-tagged Tfb1 (Tfb1.6His) and HA-tagged Kin28 (Kin28.HA) were created. Whole-cell extract (WCE) was prepared and passed over BioRex-70 and phosphocellulose columns. Two peaks of Kin28 eluted from the phosphocellulose column, although only one peak also contained Tfb1 (center blots). The two peaks were individually subjected to further purification over Ni-NTA agarose and immunoprecipitation via the HA tag on Kin28. Immunoblotting analysis was carried out to determine the composition of resulting purified complexes I and II. Both contained Kin28, Ccl1, and Tfb3. Complex II also contained Tfb1, identifying this peak as TFIIH.