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

FIG. 5.

FIG. 5.

The BAF250b complex contains an E3 ligase activity that ubiquitinates histone H2B at K120. (A) Immunopurification of Flag-BAF250b complex. A silver-stained gel of SWI/SNF purified from a HeLa cell line, 16e, induced to express Flag-BAF250b is shown (lane 2). Purification from uninduced (−) 16e cells is shown as a negative control (lane 1). The SWI/SNF subunits identified by mass spectrometry are indicated. (B) Identification of SWI/SNF subunits by immunoblotting. Endogenous BRG1, BAF170, BAF155, and E3 ligase subunit Cul2 were detected in Flag-BAF250b-immunopurified material eluted with Flag peptide from induced cells (lane 4). An antibody to Flag was used to detect Flag-BAF250b. (C) The BAF250b complex monoubiquitinates histone(s) in a ubiquitination assay. An in vitro nucleosome ubiquitination assay mixture was reconstituted with the Flag-BAF250b complex (lanes 2 to 4), control Flag-eluted material (lane 1), or recombinant Ring1b (lane 5) and HA-ubiquitin (lanes 1 to 5). The products were immunoblotted (WB) with an antibody to HA and H2B. Ten microliters of eluted material correlates roughly with each lane in the silver-stained gel shown in panel A. Oligonucleosomes were omitted from the reaction in lane 4. Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2. (D) Bacterially expressed BAF250bCTD interacts with histone H2B. GST-BAF250bCTD (HR2) (lane 2) and control GST-TAFH (lane 3) proteins were expressed and purified from E. coli and incubated with histones extracted from HeLa cells. Immunoblotting (WB) with an antibody to H2B shows specific binding of H2B to BAF250bCTD. The lower panel shows the upper portion of the same gel stained with Coomassie blue. (E) The BAF250b complex monoubiquitinates histone H2B on K120. An in vitro nucleosome ubiquitination assay was performed as for panel C with the Flag-BAF250b complex (lanes 2 and 4), control material (lane 1), or recombinant Ring1b (lanes 3 and 5). The products were immunoblotted with an antibody to HA and H2B. The nucleosomes used were reconstituted in vitro with wild-type H2B (lanes 1 to 3) or H2B with lysine-120 mutated to alanine (H2BK120A) (lanes 4 and 5). Arrow, ubiquitinated H2A/H2B; *, HA-Ub-conjugated E1 and E2.