primary antibody mouse monoclonal iggik anti cas9 ab 7a9 3a3  (Cell Signaling Technology Inc)

Journal: Nature cell biology

Article Title: Lineage-determining transcription factors constrain cohesin to drive multi-enhancer oncogene regulation

doi: 10.1038/s41556-025-01827-2

Figure Lengend Snippet: a , Heatmap displaying enrichment of active histone marks at 25,227 reproducible EBF1-binding sites determined by two ChIP-grade antibodies (RG and MP). EBF1-binding sites are ordered by hierarchical clustering of normalized EBF1, H3K27ac, H3K4me1 and H3K27me3 ChIP-seq signals. b , Western blotting of EBF1 in EBF1-FKBP-KI Granta519 clones 27 and 97 showing rapid and reproducible removal and restoration of EBF1 protein levels after dTAG V -1 (referred to as dTAG) treatment and washout, respectively. The 125 nM dTAG treatment and washout (WO) are examined for 0–24 h. β-Actin is a loading control. c , EBF1 loss impedes growth of Granta519 clone 27. Relative cell growth (CellTiter Glo) of clone 27 with or without 125 nM dTAG treatment for 6 days. Data represent mean ± s.e.m. of n = 5 biological replicates. P value obtained by two-sided t -test. d , Heatmap of EBF1 occupancy showing 7,777 bona fide EBF1-bound elements determined with log 2 (fold change) < −1 in EBF1-removed (6 h + dTAG and 24 h + dTAG versus 0 h) and log 2 (fold change) > 1 in EBF1-restored (6 h WO and 24 h WO versus 24 h + dTAG) Granta519 clone 27. Each column is centred on EBF1-bound elements ± 2-kb flanking sequences with 50-bp resolution. e , Pileup plots showing TAD boundaries are invariant in Granta519-Cas9 cells with (top) and without (bottom) EBF1 expression. Centred around 575 EBF1-bound TAD boundaries (left). 1,867 EBF1-unbound boundaries (right). f , Population-average enhancer–promoter hubs defined by EBF1 and SMC1 HiChIP in Granta519. Groups of interacting enhancers and/or promoters are plotted in an ascending order of their total pairwise interactions from EBF1 HiChIP using RG antibody (left), MP antibody (middle) and SMC1 HiChIP (right). Population-average enhancer–promoter hubs are defined as the groups of enhancers and promoters with total pairwise interactions above the elbow of total interaction distribution. The top two hubs of each HiChIP are marked and named with their representative genes. RG, a polyclonal anti-EBF1 antibody recognizing an N-terminal EBF1 peptide; MP, Millipore anti-EBF1 antibody. g , h , Gene-dense DOT1L and gene-sparse BCL2 hubs display distinct distributions of enhancers and promoters and loops across comparable linear genomic spans. ChIP-seq tracks showing enrichment of EBF1, SMC1, CTCF and H3K27ac at proximal and distal enhancers of DOT1L ( g ) and BCL2 ( h ), which are the top two highest interacting hubs defined in f . HiChIP arcs displaying normalized significant interactions among enhancers and promoters. Bottom track indicating positions of expressed Ensembl genes in Granta519. i , Box-and-whisker plots showing higher number of genes per megabase in gene-dense compared with gene-sparse hubs defined by SMC1 HiChIP. The number of hubs in each group is listed in parentheses. Box-and-whisker plots show centre line, median; box limits, upper (75th) and lower (25th) percentiles; and whiskers, 1.5 × interquartile range. P values obtained by a two-sided Wilcoxon rank-sum test. j , Barplots depicting the fraction of interactions among enhancers (E) and promoters (P) in gene-dense and gene-sparse hubs defined by EBF1 HiChIP, RG antibody. The number of hubs in each group is listed in parentheses. k , l , Gene Ontology (GO) terms specifically enriched in gene-dense ( k ) or gene-sparse ( l ) hubs. The top 500 expressed genes in each class of hubs are used as input. Source numerical data and unprocessed blots are available in source data .

Article Snippet: Primary antibodies: GAPDH (D16H11) XP (CST, cat. no. 5174); MYC (Y69) (Abcam, cat. no. ab32072); β-actin clone AC-74 (Sigma-Aldrich, cat. no. A5316); Cas9 (7A9–3A3) (CST, cat. no. 14697); and SMC1 (BL-205–2G8) (Bethyl, cat. no. A700–018).

Techniques: Binding Assay, ChIP-sequencing, Western Blot, Clone Assay, Control, Expressing, HiChIP, Whisker Assay

Journal: Nature cell biology

Article Title: Lineage-determining transcription factors constrain cohesin to drive multi-enhancer oncogene regulation

doi: 10.1038/s41556-025-01827-2

Figure Lengend Snippet: a , Box-and-whisker plots depicting higher expression (left) and essentiality (right) of EBF1 in Non-Hodgkin B lymphomas compared to T cell leukaemia/lymphoma from DepMap. The number of cell lines are indicated in parentheses. Box-and-whisker plots: centre line, median; box limits, upper (75th) and lower (25th) percentiles; whiskers, 1.5 interquartile range. b , Western blotting of EBF1 in Cas9-expressing JVM-2, Granta519 and PGA-1 MCL showing efficient depletion of EBF1 three days after transfection of sgRNA targeting EBF1. β-actin is loading control. c , d , Flow cytometry plots ( c ) and quantification ( d ) of cell apoptosis and death measured by Annexin V and ToPro-3 staining. Ctrl and EBF1-KO Cas9-expressing MCL cells were sorted three days post lentiviral transduction and cultured for three days. See Supplementary Fig. 1 for gating strategy. Data represent mean ± S.D. of 3 replicates per condition. P value: two-sided t-test. e , Cumulative distribution plot (CDF) of distances between each reproducible EBF1 peak to the closest expressed gene TSS in Granta519 showing > 75% EBF1 peaks are located 10 kb away from TSS. f , Schematics of knocking in FKBP F36V domain at the stop codon of endogenous EBF1 gene in Granta519-Cas9 cells. Single cell clones were selected with blasticidin and successful insertion of the FKBP F36V cassette was validated with genomic DNA PCR. Clones with efficient EBF1 degradation after dTAG V -1 treatment (referred to as dTAG) are used. g , Hierarchical clustering of normalized reads (RPKM) from Ctrl and EBF1-KO Granta519-Cas9, and EBF1-FKBP-KI clones 26, 27, 29, 97 with 0, 6, 24-h 125 nM dTAG treatment showing higher similarity of clones 27 and 97 with Granta519-Cas9 transcriptome. h , Volcano plot showing ATAC-seq signal fold enrichment (x axis) versus false discovery rate (FDR) (y axis) in Granta519-EBF1-FKBP-KI clone 27 treated with 125 nM dTAG for 6, 24, 48, 72 h compared with untreated cells. Each point depicts an accessible element, colour coded by blue, red, and black based on significantly decreased, increased, or unchanged accessibility in treated cells, respectively. Significance cutoff: FDR < 1E-5 and Log2(fold change) => 1. i , Heatmaps displaying overall unchanged normalized accessibility levels at dynamic EBF1-binding sites. Each column of ATAC-seq signals is centred on bona fide EBF1-bound elements per Fig. 1d ± 2 Kb flanking sequences with 50 bp resolution. j , Scatterplot of PC1 values of Hi-C contact matrices in Ctrl and EBF1-KO Granta519-Cas9 cells showing largely invariable A/B compartments. k , Box-and-whisker plots of EBF1 (left), YY1 (middle) and H3K27ac (right) loading at the promoters (P) and enhancers (E) interacting with significant long-range DNA loops of SMC1 HiChIP in Granta519. EBF1 preferentially binds enhancers, while YY1 preferentially binds promoters. H3K27ac is negative control. EE: n = 11,818; PE: n = 10,739; PP: n = 3,806. Box-and-whisker plots: see panel a . FWER-adjusted p values: two-side Wilcoxon rank-sum test. l , Venn diagram comparing overlapping of the genomic coordinates of enhancer–promoter hubs in Granta519 SMC1 and EBF1 (RG and MP) HiChIP showing high concordance of hubs identified by three assays. m , Cumulative distribution plots showing expressed genes count in each enhancer–promoter hub defined by EBF1 (RG and MP) and SMC1 HiChIP. n , Box-and-whisker plots showing higher number of genes per megabase in gene-dense compared to gene-sparse hubs defined by EBF1 HiChIP using RG (left) and MP (right) antibodies. The number of hubs in each group is listed in parentheses. Box-and-whisker plots: see panel a . P values: two-sided Wilcoxon rank-sum test. Source numerical data and unprocessed blots are available in Source data .

Article Snippet: Primary antibodies: GAPDH (D16H11) XP (CST, cat. no. 5174); MYC (Y69) (Abcam, cat. no. ab32072); β-actin clone AC-74 (Sigma-Aldrich, cat. no. A5316); Cas9 (7A9–3A3) (CST, cat. no. 14697); and SMC1 (BL-205–2G8) (Bethyl, cat. no. A700–018).

Techniques: Whisker Assay, Expressing, Western Blot, Transfection, Control, Flow Cytometry, Staining, Transduction, Cell Culture, Clone Assay, Binding Assay, Hi-C, HiChIP, Negative Control

Journal: bioRxiv

Article Title: A vesicle-based platform for high-efficiency, high-viability CRISPR/Cas9 knockout in primary human myeloid cells

doi: 10.64898/2025.12.28.696771

Figure Lengend Snippet: (A)Schematic workflow. After isolation, CD14 + monocytes are seeded in the desired well format and treated with vesicles. Cytokines are added to the media to induce monocytic differentiation. After differentiation, gene editing is evaluated by flow cytometry and genomic sequencing. (B) Representative flow cytometry plots showing B2M knockout in vesicles-treated CD14 + monocytes. (C) B2M knockout efficiency in monocyte-derived macrophages (MDMs) treated with increasing concentrations of EVs or VLPs carrying Cas9 fused to MLV Gag, compared with nucleofection. B2M expression was measured by flow cytometry 7 days post-treatment. Data are mean ± SEM (n=3 donors). Statistical analysis: two-way ANOVA. (D) Cell viability of CD14 + cells following EV/VLP treatment or nucleofection (as in C), assessed by CellTiter-Glo. Data are mean ± SEM (n=3 donors). (E) TNF-α production by MDMs after EVs, VLPs, nucleofection, or LPS stimulation (positive control), quantified by ELISA on supernatants collected after overnight incubation. Data are mean ± SEM (n=3 donors). Statistical analysis: two-way ANOVA. (F) B2M knockout efficiency in SUP-T1 cell lines treated with equal volumes of EVs produced with decreasing VSV-G levels. The percentage of VSV-G indicates the fraction of the VSV-G plasmid relative to the total DNA used for transfection. B2M expression was analysed by flow cytometry 5 days post-treatment. One representative experiment out of 3 is shown (n=3)(G) Viability analysis for the experiment shown in F. One representative experiment out of 3 is shown (n=3)(H) Functional editing in primary MDMs assessed by flow cytometry 7 days after treatment with VLPs high (10%) or low VSV-G (0.5%). Data are mean ± SEM (n=3 donors). Statistical analysis: Two-way ANOVA. (I) Cell viability for the experiment shown in H. Data are mean ± SEM (n=3 donors). Statistical analysis: Two-way ANOVA. *P ≤ 0.05; **P ≤ 0.01;***P ≤ 0.001

Article Snippet: Membranes were blocked in 5% milk in TBS-T and probed with an anti–S. pyogenes Cas9 antibody (Cell Signaling; clone 7A9-3A3) followed by HRP-conjugated anti-mouse secondary antibody.

Techniques: Isolation, Flow Cytometry, Genomic Sequencing, Knock-Out, Derivative Assay, Expressing, Positive Control, Enzyme-linked Immunosorbent Assay, Incubation, Produced, Plasmid Preparation, Transfection, Functional Assay