Journal: eLife

Article Title: LIN37-DREAM prevents DNA end resection and homologous recombination at DNA double-strand breaks in quiescent cells

doi: 10.7554/eLife.68466

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-BARD1 (Rabbit polyclonal) , Thermo Fisher Scientific , PA5-85707 , WB (1:1000).

Techniques: Recombinant, Plasmid Preparation, Genome Wide, CRISPR, Clone Assay, Flow Cytometry, Staining, Sequencing, Software, Microscopy

Journal: Cell reports

Article Title: SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation

doi: 10.1016/j.celrep.2021.108921

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-BARD1 , Bethyl , Cat# A300-263A; RRID: AB_2061250.

Techniques: Recombinant, Software

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 1 p50 binds the BARD1 BRCT domains. a Silver stained gel of nuclear extract from 293T cells transfected with empty vector (EV), HA-p50wt or HA- p50S328A. Immunoprecipitation (IP) was performed with anti-HA. Asterisk indicates exogenous p50. Arrowhead, band analyzed by MS/MS. b Reciprocal Co-IP in 293T cells expressing S-tag BARD1 and FLAG-p50. IP with anti-FLAG (left) or S-agarose (right). Immunoblot (IB) with the indicated antibody. c Co-IP in primary wild type (WT) MEFs following IP of endogenous protein and IB with indicated antibodies. d Co-IP in WT MEFs treated with vehicle or HU (2 mM, 4 h). IP with anti-p50 and IB with anti-Bard1. e WT MEFs expressing TopBP1ER or GFPER were treated with tamoxifen (TAM, 4 h). IP with anti- p50 and IB with anti-Bard1 or indicated antibody. Inset: schematic of TopBP1ER or GFPER. f Schematic representation of BARD1 (ANK, ankyrin repeat). g Co- IP in 293T cells transfected with FLAG-BARD1 deletion constructs (FL, full length; N1-202, amino acids 1-202) and HA-p50. IP performed with anti-FLAG and IB with anti-HA. h Nickel column purification of 6xHis-p50 incubated with GST-BRCT or GST alone. Eluted proteins were examined with anti-p50 antibody. Input sample was probed with anti-p50 or anti-GST. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Staining, Transfection, Plasmid Preparation, Immunoprecipitation, Tandem Mass Spectroscopy, Co-Immunoprecipitation Assay, Expressing, Western Blot, Construct, Nickel Column, Incubation, Control

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 2 BARD1 interacts with a conserved p50 phospho-serine motif. a Putative BARD1 BRCT-binding motif. Motif in human p50 is underlined. Sequence of p50 mutants (lower). b 293T cells expressing FLAG-BARD1 and the indicated HA-p50 construct. IP and IB performed with the indicated antibodies. c 293T cells expressing TopBP1ER or GFPER transfected with p50wt (left) or p50S337A (right) and treated with vehicle or TAM (4 h). IP with anti-HA and IB with anti-phospho-p50-S337 antibody. d 293T cells expressing TopBP1ER were transfected with si-CHK1 or si-control and treated with vehicle or TAM. IB was performed with the indicated antibody. e Kinase assay with bacterially expressed p50wt or the indicated p50 mutant and recombinant CHK1. Autoradiogram (32P) and IB with indicated antibody of the same membrane. Arrow, phosphorylated p50. f Co-IP in primary WT MEFs expressing TopBP1ER

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Binding Assay, Sequencing, Expressing, Construct, Transfection, Control, Kinase Assay, Mutagenesis, Recombinant, Membrane, Co-Immunoprecipitation Assay

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 3 BARD1 binding promotes p50 mono-ubiquitination. a Immunoblot (IB) in 293T cells expressing TopBP1ER transfected with Myc-ubiquitin (Myc-Ub) and S-tag p50wt treated with tamoxifen (TAM) for the indicated time. IP performed with S-agarose and IB with anti-Myc antibody. b Cells transfected as in A and si-BARD1 or si-control treated with TAM (4 h). Nuclear fractions were isolated and IP and IB performed as in A. H3, Histone H3. c Bard1-null mouse cells transfected with Myc-Ub, S-tag p50wt and FLAG-BARD1. IP with S-agarose and IB with anti-Myc antibody. d Bard1-null cells transfected with FLAG- BARD1, S-tag p50wt, and Myc-0KUb. IP with S-agarose and IB with anti-Myc antibody. e Bard1-null cells transfected with FLAG-BARD1, Myc-Ub, S-tag p50wt, or p50S337A. IP performed with S-agarose and IB with anti-Myc antibody. f Bard1-null cells transfected with FLAG-BARD1, Myc-Ub, and S-tag p50wt

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Binding Assay, Ubiquitin Proteomics, Western Blot, Expressing, Transfection, Control, Isolation

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 4 p50 is mono-ubiquitinated at K354 and/or K356. a Co-IP in 293T cells transfected with Myc-Ub and the indicated S-tag p50 deletion construct. IP with S-agarose and IB with anti-Myc antibody. b Alignment of p50 sequences. Potential human mono-Ub sites underlined. c 293T cells transfected with Myc-Ub, S-tag p50wt, or the indicated p50 mutant. 2KR, K354R/K356R. IP with S-agarose and IB with anti-Myc antibody. d Bard1-null cells transfected with FLAG-BARD1, Myc-Ub, S-tag p50wt, or p502KR. IP with S-agarose and IB with anti-Myc antibody. e 293T cells transfected with Myc-Ub, S-tag p50wt or p502KR, and treated with HU (2 mM, 4 h). IP with S-agarose and IB with anti-Myc antibody. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Co-Immunoprecipitation Assay, Transfection, Construct, Mutagenesis, Control

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 5 Mono-ubiquitination stabilizes p50 and decreases chromatin recruitment. a Bard1-null cells transfected with BARD1 or empty vector (EV) and the indicated S-tag p50 construct. IB performed with anti-S antibody or anti-GAPDH at the indicated time after cycloheximide (CHX) addition. See Supplementary Fig. 4a for quantification. b 293T cells infected with sh-RNA targeting BARD1. IB performed with indicated antibody. c Bard1-null cells transfected with BARD1 and the indicated S-tag p50 construct fused with a C-terminal Ubiquitin (0 K and GG/AA mutant Ub). IB performed at the indicated time after CHX addition. See Supplementary Fig. 4b for quantification. d 293T cells transfected with the indicated p50 construct were separated into nuclear (nuc) and cytoplasmic (cyto) fractions. IB with anti-S antibody or indicated loading control. e 293T cells transfected with S-tag p50wt, p502KR, or p502KR-Ub were fractionated into cytoplasmic (cyto) fraction. Twenty percent of the cell pellet was isolated for analysis of whole-cell extract (WCE). IB was performed with anti-S tag. f 293T cells transfected with Myc-Ub were separated into nuc and cyto fractions. IP in each fraction performed with anti- p50 antibody or anti-IgG and IB with anti-Myc. g Electrophoretic mobility shift assay (EMSA, upper) or immunoblot (IB, lower) of increasing amounts of bacterially expressed p50wt, or p50 mutant, protein using the IgK probe. The same membrane was analyzed by autoradiography (32P) and IB. h EMSA (upper) or IB (lower) of p50wt (WT) and p50S337D (D) protein using the κB-site probe from the indicated gene (tumor necrosis factor alpha, TNFα; histone 2B, H2B). IB of input proteins demonstrates equal loading. IgK−1A is the IgK with adenine at the –1 position. S and N, specific and non-specific competitor, respectively. i DNA motifs most significantly enriched for p50 on ChIP-Seq analysis of 293T cells expressing TopBP1ER treated with vehicle. j Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of p50 ChIP-seq peaks at baseline ranked by –log10 (P-value). k ChIP-seq analysis of p50- binding peaks. Heatmap (left) and histogram (right) for average peak intensity was plotted for ChIP-Seq data from vehicle and tamoxifen (TAM) treated samples. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Construct, Infection, Mutagenesis, Control, Isolation, Electrophoretic Mobility Shift Assay, Western Blot, Membrane, Autoradiography, ChIP-sequencing, Expressing, Binding Assay

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 6 p50 mono-ubiquitination regulates cell cycle progression. a Synchronized primary WT MEFs were harvested at the indicated cell cycle stage and nuclear fractions isolated. IB using anti-p50-pS337, anti-p50, and anti-Cyclin E. b Co-IP in primary WT MEFs synchronized and released as in a. IP with anti-p50 or IgG and IB with anti-Bard1 or indicated antibody. c 293T cells transfected with Myc-Ub and S-tag p50wt were harvested at the indicated cell cycle stage and nuclear extract used for IP with S-agarose followed by IB with anti-Myc or anti-BARD1 antibody. d 293T cells transfected with Myc-Ub and p50 constructs were harvested at S phase. IP of nuclear fractions with S-agarose and IB with anti-Myc or anti-BARD1. e Immortal Nfkb1-/- MEFs stably expressing empty vector (EV), p50wt (WT), or p502KR (2KR) were pulsed with 5-ethynyl-2 deoxyuridine (EdU) and positive staining quantified. Data show mean value from n = three biologically independent experiments, ± SEM. *P = 0.012, **P = 0.004, two-sided Student’s t-test. Inset: IB with anti-p50. f Immortal Nfkb1-/- MEFs stably expressing EV or the indicated p50 mutant were plated and cell number counted every 24 h. Data show mean number from three biologically independent experiments, ± SEM. **P < 0.001, EV vs. WT, two-sided Student’s t-test. g Immortal Nfkb1-/- MEFs stably expressing the indicated construct were injected into flanks of nude mice and tumors harvested at 2 weeks. Representative experiment (left panel, n = 5 animals). Box plot: center-line is median, box limits represent upper and lower quartiles and whiskers are minimum and maximum. **P < 0.001, two-sided Student’s t- test. h Immortal Nfkb1-/- MEFs stably expressing EV, p50wt, p502KR, or p502KR-Ub (2KR-Ub) were pulsed with EdU and positive staining quantified. Data show mean value from two biologically independent experiments. i Venn diagram illustrating overlap between genes associated with p50-binding peaks in ChIP-Seq analysis from Fig. 5 and periodically regulated genes from Cyclebase 3.0. j ChIP-seq analysis of p50-binding peaks. Heatmap (left) and histogram (right) for average peak intensity was plotted for ChIP-Seq data from cells synchronized at G1 and S phase. k ChIP-qPCR of p50 binding to the CCNE1 promoter in 293T cells at the indicated cell cycle phase. Data show mean enrichment of p50 relative to input and IgG control, ± SEM. from n = 3 biologically independent experiments.**P < 0.001, two-sided Student’s t-test. l IB using Nfkb1-/- MEFs expressing EV, S-p50wt or S-p502KR using anti-Cyclin E and anti-S tag. All blots are representative of at least two biologically independent experiments. Analysis of fold-change normalized to control lane shown below IB where indicated.

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Ubiquitin Proteomics, Isolation, Co-Immunoprecipitation Assay, Transfection, Construct, Stable Transfection, Expressing, Plasmid Preparation, Staining, Mutagenesis, Injection, Binding Assay, ChIP-sequencing, ChIP-qPCR, Control

Journal: Nature communications

Article Title: p50 mono-ubiquitination and interaction with BARD1 regulates cell cycle progression and maintains genome stability.

doi: 10.1038/s41467-020-18838-2

Figure Lengend Snippet: Fig. 7 BARD1 and p50 protein in human cancer. a Analysis of breaks and gaps in primary Nfkb1-/- MEFs expressing either p50wt or p502KR. Cells were treated with vehicle or aphidicolin (APH, 0.1 μM 24 h). Data show gaps or breaks from 25 random metaphase cells, ± SEM. *P = 0.003, **P = 0.007, two- sided unpaired Student’s t-test. Representative metaphase spreads shown (left). b Co-IP in Bard1-null mouse cells transfected with Myc-Ub, S-tag p50wt

Article Snippet: The following primary antibodies were used: anti-FLAG M2 antibody (1:20,000, Sigma-Aldrich, F1804); anti-FLAG rabbit monoclonal antibody (1:10,000, Cell Signaling, #14793), Anti-HA tag antibody ChIP Grade (1:20,000, Abcam, Ab9110); anti-S-Tag antibody (1:10,000, BioLegend, 688102); anti-Myc tag antibody (1:5,000, Santa Cruz, sc-40); anti-BARD1 mouse monoclonal antibody (1:500, Santa Cruz, sc-74559); anti-BARD1 rabbit polyclonal antibody (1:2,000, Bethyl Laboratories, A300-263A); anti-BARD1 rabbit polyclonal antibody (1:1,000, Santa Cruz, sc11438); anti- NFκB1/p50 mouse monoclonal antibody (1:500, Santa Cruz, sc8414X); anti-BCL-3 antibody (1:500, Santa Cruz, sc-185); anti- cyclin E antibody (1:2,000, Santa Cruz, sc-198); anti- BRCA1 antibody (1:500, Santa Cruz, sc-6954); anti- Chk1 antibody (1:2,000, Santa Cruz, sc-8408); anti-GAPDH antibody (1:20,000, Santa Cruz, sc-32233); anti-Histone H3 antibody (1:50,000, Santa Cruz, sc-517576).

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection