FIG. 1.

GAS41 is a core subunit of the human TIP60 and SRCAP complexes. (A) Coimmunoprecipitation of GAS41 with core subunits of the TIP60 complex. 293T cells were transfected with empty vector or vector expressing FLAG-GAS41, and whole-cell lysates were immunoprecipitated with M2 agarose beads. After extensive washes, the captured FLAG-GAS41 was eluted with FLAG peptide under nondenaturing conditions. Eluates were analyzed by immunoblotting and HAT assays as indicated. Histone H2A was not separated from H3 in this minigel in which acetylated H2A partially overlapped with H3 (see Fig. 4B for better separation). (B) Sepharose CL6B gel filtration chromatography. Nuclear extracts were prepared from 293T cells stably expressing FLAG-GAS41 and directly applied to the Sepharose CL6B gel filtration column. Each fraction (0.1%) was analyzed by immunoblotting for TRRAP and GAS41. Fractions corresponding to the TRRAP peak (fractions 4 and 5) were combined and subjected to M2 agarose affinity purification. After being washed, captured FLAG-GAS41 was sequentially eluted with FLAG peptide and 0.1 M glycine (pH 2.5) and analyzed by immunoblotting for TRRAP, TIP60, p400, H2AZ, and GAS41.