American Society of Hematology

Figure 1

$Figure 1. Leukemic BAF complexes are assembled around BRG ATPase. (A) Strategy for purification and sequencing of BAF complexes expressed in leukemia. Highly stringent conditions were used to isolate only the core components of the complexes and tightly associated factors. See supplemental Table 1 for all identifications. (B) Immunoprecipitation and western blot analysis of BRG/BRM ATPase subunits in nuclear extracts isolated from FLA2 leukemia using anti-BRG/BRM (J1) antibodies, which recognize both ATPases with equal efficiency. (C) Silver-stain analysis of immunopurified (anti-BRG/BRM) BAF complexes in nuclear extracts isolated from FLA2 leukemia. (D) Subunit composition of leukemic BAF (leukBAF) complexes identified by a proteomics approach in FLA2 and FLB1 leukemia. Frequencies of leukemia-initiating cells are 1 in 1.4 and 1 in 347, respectively. †Total number of different peptides for each protein entry. ‡Number of specific peptides corresponding to a unique protein entry in IPI mouse database. §Sequence coverage (%). •Mascot score with false discovery rate <2%. (E) Relative abundance of Brg and Brm messenger RNAs (mRNAs) in FLA2 and FLB1 leukemia based on adjusted reads by coverage obtained by RNA-seq.32 (F) Relative abundance of BRG and BRM ATPases in nuclear extracts isolated from various mouse A9M-derived leukemias using anti-BRG and BRM specific antibodies. BAF60B is a loading control. HEK293 cells were used as a positive control for BRM expression. (G) Schematic representation of leukBAF complexes assembled on BRG ATPase. Complexes are drawn in a jigsaw puzzle configuration to denote the apparent fit of the subunits within the complexes. These positions have not been experimentally defined, except for actin and BAF53, which contact the catalytic domain of BRG. Subunits shown in dashed outline are inconstant components of the complexes. Domains that bind DNA or modified histones and hence could target the complexes to specific loci independent of (or in cooperation with) transcription factors are shown. 2DLC-MS/MS, two-dimensional liquid chromatography-tandem mass spectrometry; Ub, unbound fraction; IgG, immunoglobulin; E-1 and −2, elution 1 and 2, respectively. See also supplemental Figures 1-3 and supplemental Tables 1-2.$

Leukemic BAF complexes are assembled around BRG ATPase. (A) Strategy for purification and sequencing of BAF complexes expressed in leukemia. Highly stringent conditions were used to isolate only the core components of the complexes and tightly associated factors. See supplemental Table 1 for all identifications. (B) Immunoprecipitation and western blot analysis of BRG/BRM ATPase subunits in nuclear extracts isolated from FLA2 leukemia using anti-BRG/BRM (J1) antibodies, which recognize both ATPases with equal efficiency. (C) Silver-stain analysis of immunopurified (anti-BRG/BRM) BAF complexes in nuclear extracts isolated from FLA2 leukemia. (D) Subunit composition of leukemic BAF (leukBAF) complexes identified by a proteomics approach in FLA2 and FLB1 leukemia. Frequencies of leukemia-initiating cells are 1 in 1.4 and 1 in 347, respectively. ^†Total number of different peptides for each protein entry. ^‡Number of specific peptides corresponding to a unique protein entry in IPI mouse database. ^§Sequence coverage (%). •Mascot score with false discovery rate <2%. (E) Relative abundance of Brg and Brm messenger RNAs (mRNAs) in FLA2 and FLB1 leukemia based on adjusted reads by coverage obtained by RNA-seq.³² (F) Relative abundance of BRG and BRM ATPases in nuclear extracts isolated from various mouse A9M-derived leukemias using anti-BRG and BRM specific antibodies. BAF60B is a loading control. HEK293 cells were used as a positive control for BRM expression. (G) Schematic representation of leukBAF complexes assembled on BRG ATPase. Complexes are drawn in a jigsaw puzzle configuration to denote the apparent fit of the subunits within the complexes. These positions have not been experimentally defined, except for actin and BAF53, which contact the catalytic domain of BRG. Subunits shown in dashed outline are inconstant components of the complexes. Domains that bind DNA or modified histones and hence could target the complexes to specific loci independent of (or in cooperation with) transcription factors are shown. 2DLC-MS/MS, two-dimensional liquid chromatography-tandem mass spectrometry; Ub, unbound fraction; IgG, immunoglobulin; E-1 and −2, elution 1 and 2, respectively. See also supplemental Figures 1-3 and supplemental Tables 1-2.

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