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. 2010 Jan 19;30(7):1673–1688. doi: 10.1128/MCB.00540-09

FIG. 1.

FIG. 1.

FIG. 1.

BAF250 contains a BC box and associates with Elo C. (A) Schematic diagram of BAF250b (GenBank accession no. AF521671). 16e, amino acids 419 to 2165; NTD, 419 to 1547; CTD, 1518 to 2165. Putative BC boxes identified by MAST scanning of human BAF250a/b and Drosophila Osa are shown. Amino acid numbering in the second column corresponds to the GenBank accession numbers listed in parenthesis. Sequences of consensus and putative BC boxes are listed in the third column, with key residues highlighted in bold. (B) The BAF250a/b C-terminal domain associates with Elo C. Upper two panels, cells were transiently transfected with Flag-Elo C alone (lanes 1, 3, and 5) or together with T7-BAF250aCTD (amino acids 1635 to 2285, GenBank accession no. O14497) (lanes 2, 4, and 6). Lysates were immunoprecipitated (IP) with α-T7 antibody, washed in a buffer containing 0.3 M KCl (lanes 3 and 4) or 0.5 M KCl (lanes 5 and 6), and analyzed by immunoblotting with an antibody to T7 or Flag. Lower two panels, the same experiment was performed with T7-BAF250bCTD. (C) Bacterially expressed Elo C interacts with BAF250b CTD. Polyhistidine (His)-tagged Elo C was expressed in E. coli, and the lysate was mixed with purified GST-BAF250bCTD (HR2) (lane 3) or control GST-TAFH (lane 2). Immunoblotting (WB) with an antibody to the poly-His tag shows specific binding of Elo C to BAF250bCTD. HR2 represents the second conserved region in BAF250bCTD, which contains the BC box (residues 1786 to 2165, boxed in gray in Fig. 1A). (D) BAF250bCTD interaction with Elo C is BC box dependent. Flag antibody immunoprecipitates of transiently transfected Flag-Elo C with T7-BAF250bNTD, T7-BAF250bCTD, or T7-BAF250bCTD with the BC box mutation (BC*) were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag. *, nonspecific band. (E) BAF250b interacts with Elo C in a BC box-dependent manner. T7 antibody immunoprecipitates of transiently transfected vector, T7-BAF250b, or T7-BAF250bBC* with Flag-Elo C were washed in a buffer containing 0.3 M KCl and immunoblotted with an antibody to T7 or Flag.