Journal: Science Advances

Article Title: Nonchromatin regulatory functions of the histone variant H2A.B in SWI/SNF genomic deposition

doi: 10.1126/sciadv.adx1568

Figure Lengend Snippet: ( A ) An N-terminal sequence alignment of H2A.B orthologs across various eutherian mammals. The N terminus of human H2A is also shown. Conserved residues R7, S9, and R15 are highlighted in red, while residues identical to human H2A.B are shaded in gray. ( B ) H2A.B PTMs identified in L1236 and L428 cells. These include arginine monomethylation (me), arginine asymmetric dimethylation (me2a), and serine phosphorylation (ph). ( C ) Western blot analyzing H2A.B immunoprecipitates using anti-sDMA and anti-aDMA antibodies. ( D ) Western blot analyzing R7me2a, R15me2a, and S9ph modifications in H2A.B immunoprecipitates using custom-made modification-specific antibodies. ( E ) Western blot analyzing PRMT1 protein levels in nontargeting control (NTC) and PRMT1 shRNA–transduced L1236 and L428 cells. β-Actin served as a loading control. The levels of PRMT1, normalized to β-actin, were quantified and displayed below the blot. ( F ) Western blot analyzing R7me2a levels in H2A.B immunoprecipitates from NTC or PRMT1 shRNA–transduced L1236 and L428 cells. The levels of R7me2a were normalized to total H2A.B and displayed below the blot. ( G ) Proximity-dependent BioID cellular assay confirming that the interaction between PRMT1 and H2A.B. L1236 cells (right panel) and L428 cells (left panel) was transduced with either an empty vector (vector-only), a BirA*-H2A.B vector, or an H2A.B-BirA* vector. Following biotin labeling, streptavidin affinity purification of cell lysates was analyzed by Western blot using an anti-PRMT1 antibody. The positive control was loaded as 1% (v/v) input. The Western blot signal for H2A.B runs below 15 kDa (the molecular weight of H2A.B is 12.7 kDa). The asterisk (*) designates non-H2A.B antibody binding.

Article Snippet: Primary antibody incubation was performed overnight at 4°C in 1% BSA in PBST using the following antibodies: H2A.B, H2A.BR7me2a, H2A.BR15me2a and H2A.BS9ph (custom-made, GenScript Biotech), SDMA (Cell Signaling Technology, 13233S), ADMA (Cell Signaling Technology, 13522S), PRMT1 (Merck Millipore, 07-404), PRMT6 (Cell Signaling Technology, 14641S), SMARCC1 (Cell Signaling Technology, 11956S; Proteintech, 17722-1-A), SMARCE1 (abcam, ab131328), TRA2B (abcam, ab31353), SMARCD1 (abcam, ab224229; Santa Cruz, sc-135843), BRG1 (Santa Cruz, sc-17796x; abcam, ab4081), BRM (Cell Signaling Technology, 11966; abcam, ab15597), ARID1A (Santa Cruz, sc-373784x), ARID2 (Santa Cruz, sc-166117x), DPF2 (Bethyl Laboratories, A303-595A), PHF10 (Thermo Fisher Scientific, PA5-30678; Invitrogen, PA5-30678), β-actin (Cell Signaling Technology, 8457S), and H2A.Z (Active Motif, 39943).

Techniques: Sequencing, Phospho-proteomics, Western Blot, Modification, Control, shRNA, Transduction, Plasmid Preparation, Labeling, Affinity Purification, Positive Control, Molecular Weight, Binding Assay

Journal: Science Advances

Article Title: Nonchromatin regulatory functions of the histone variant H2A.B in SWI/SNF genomic deposition

doi: 10.1126/sciadv.adx1568

Figure Lengend Snippet: ( A ) H2A, H2A.Z, H2A.B, H2A.BR7me2a, H2A.BR7me, or H2A.BR15me biotin-tagged N-terminal peptides were incubated with purified recombinant CBAF (BRG1 and BRM) or PBAF (BRG1 and BRM). Peptide-bound hSWI/SNF complexes were subjected to a Western blot analysis, and relative peptide binding affinities were determined using a combination of different common and subunit-specific CBAF and PBAF antibodies (see Materials and Methods). As an additional control, biotinylated recombinant human nucleosomes were also examined as an hSWI/SNF binding substrate. Error bars represent the standard error of the mean. ( B ) Western blots analyzing the levels of CN H2A.B coimmunoprecipitated BRG1 and SMARCC1 from NTC or PRMT1 shRNA–transduced L428 cells.

Article Snippet: Primary antibody incubation was performed overnight at 4°C in 1% BSA in PBST using the following antibodies: H2A.B, H2A.BR7me2a, H2A.BR15me2a and H2A.BS9ph (custom-made, GenScript Biotech), SDMA (Cell Signaling Technology, 13233S), ADMA (Cell Signaling Technology, 13522S), PRMT1 (Merck Millipore, 07-404), PRMT6 (Cell Signaling Technology, 14641S), SMARCC1 (Cell Signaling Technology, 11956S; Proteintech, 17722-1-A), SMARCE1 (abcam, ab131328), TRA2B (abcam, ab31353), SMARCD1 (abcam, ab224229; Santa Cruz, sc-135843), BRG1 (Santa Cruz, sc-17796x; abcam, ab4081), BRM (Cell Signaling Technology, 11966; abcam, ab15597), ARID1A (Santa Cruz, sc-373784x), ARID2 (Santa Cruz, sc-166117x), DPF2 (Bethyl Laboratories, A303-595A), PHF10 (Thermo Fisher Scientific, PA5-30678; Invitrogen, PA5-30678), β-actin (Cell Signaling Technology, 8457S), and H2A.Z (Active Motif, 39943).

Techniques: Incubation, Purification, Recombinant, Western Blot, Binding Assay, Control, shRNA