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. 2022 Oct 26;13:6384. doi: 10.1038/s41467-022-34200-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic of mouse brain soluble nuclear protein extract (NE) preparation, density sedimentation of nuclear proteins over a 10–30% glycerol gradient, and immunoprecipitation of BAF chromatin remodeling complexes. Blue lettering indicates neuronal-specific BAF subunits. Red lettering indicates PBAF-specific subunits. b Density sedimentation of adult Brwd1^FLAG-HA brain NE over a 10–30% glycerol gradient indicates that BRWD1 predominantly associates with large protein complexes. Subunits of BAF and AP-1 complexes serve as molecular weight markers: SMARCA2/4 antibody indicates all BAF complexes including non-canonical GBAF (~1 MDa)⁷⁵, canonical BAF (~2 MDa) and Polybromo-containing BAF (PBAF, ~3 MDa); ACTL6B and SS18L1 indicate neuronal-specific BAF complexes; c-Jun indicates AP-1 (160–440 kDa). HA signal at the expected molecular weight of BRWD1-FLAG-HA (~260 kDa) is observed in fractions containing the BAF complex. c Endogenous BRWD1-FLAG-HA interacts with BAF complexes in embryonic brain. BAF complexes were immunoprecipitated from Brwd1^FLAG-HA brain NE with antibodies against the BAF core ATPase SMARCA4, the neural progenitor subunit SS18, the neuronal subunit SS18L1 or IgG as a control. Endogenous BRWD1-FLAG-HA robustly co-immunoprecipitated with SMARCA4 and the neural progenitor subunit SS18, but less so with the neuronal subunit SS18L1 from E17.5 brain. d BAF complexes purified from adult Brwd1^FLAG-HA brain NE with antibodies against SMARCA4 or the neuronal subunit SS18L1 co-immunoprecipitate BRWD1-FLAG-HA. e The stability of the BAF:BRWD1-FLAG-HA interaction was challenged with increasing concentrations (0.25-4 M) of the denaturing agent, urea. A fraction of BRWD1 remained bound to BAF in up to 4 M urea, surpassing the stability of the dedicated BAF subunit, SMARCB1. f Quantification of urea denaturation experiments, as shown in e, with the amount of bound protein normalized to the amount of immunoprecipitated SMARCA4 (n = 3 experiments). Source data are provided as a source data file. See Supplementary Fig. 15 for uncropped blots with MW markers.