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. 2002 Oct 1;16(19):2509–2517. doi: 10.1101/gad.992102

Figure 2.

Figure 2

BAF53b is a subunit of a brain-specific chromatin remodeling complex. (A) BAF53b mRNA expression is restricted to the nervous system. Expression patterns for BAF53b (top) and BAF53a (bottom) in different adult mice tissues using ribonuclease protection assay. Arrows, protected fragments for both RNAs. Note that the faint upper band in all lanes of the BAF53b gel is the undigested probe. (B) BAF53b protein is expressed exclusively in mouse brain nuclear extract and reciprocally immunoprecipitates with the BAF ATPase subunit BRG1. Western blots for BAF53b (top) and BRG1 (bottom) using anti-BAF53b or anti-BRG1 antibodies. (Lanes 1–3) Nuclear extracts (10 μg) prior to purification. Immunoprecipitations using the antibodies shown on top of each panel are in lanes 4–7. (C) The bBAF complex is unique. (Top) Silver-stained SDS-PAGE gel (7.5%) showing immunopurified chromatin remodeling complexes from mouse brain nuclear extracts (Brain, lane 1) or HeLa cells (lane 3) using anti-BRG1 antibodies. (Lane 4) Immunopurification control of brain extracts with 12CA5 antibodies. Molecular weight markers (MW, lane 2) are indicated at the right. BAF subunits are shown at the left. Asterisks indicate nonspecific proteins. IgG is immunoglobulin heavy chain. (Bottom) BAF53b and BAF53a are mutually exclusive subunits of BAF chromatin remodeling complexes. After immunopurification of BAF complexes with the indicated antibodies, blots were analyzed with BAF53b- and BAF53a-specific antibodies. (Lane 3) Control immunoprecipitation using 12CA5 antibody. (Lanes 4,5) Input nuclear extracts [HeLa (He); Brain (Br); 10 μg]. (D) bBAF has ATP-dependent mononucleosome disruption activity. bBAF complexes attached to beads were immunopurified as described above and assayed on mononucleosomes in the absence (lane 7) or presence (lane 8) of 1 mM ATP. As a control, BAF complexes from Jurkat cells were used (lanes 5,6). No disruption is observed in the absence of chromatin remodeling complexes (lanes 3,4). Arrows show the ATP-dependent disruption of mononucleosomes (cf. lanes 5–8 and 3,4). Lanes 1 and 2 show controls for naked DNA and mononucleosomes alone, respectively.