anti h2bub  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: NAD + sensing by PARP7 regulates the C/EBPβ-dependent transcription program during adipogenesis

doi: 10.1016/j.celrep.2026.116929

Figure Lengend Snippet: (A) Western blot showing a time course of PARP7 and C/EBPβ expression in 3T3-L1 cells differentiated with MDI cocktail. (B) Bar graphs showing the enrichment of C/EBPβ at the promoters of target genes Pparg , Atf3 , and Klf15 , as assayed by ChIP-qPCR, in 3T3-L1 cells with or without differentiation using MDI cocktail. Mean + SEM; n = 3. Asterisks indicate significant differences from control; ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. (C) IP-western assays showing interactions between C/EBPβ and PARP7 in 3T3-L1 cells. 3T3-L1 cells with Dox-inducible ectopic expression of FLAG-C/EBPβ were subjected to FLAG IP. The IPs were subjected to western blotting for FLAG and PARP7. (D) Western blots of chromatin fractions from 3T3-L1 cells showing two p300-mediated histone modifications, H3K27ac and H2BK5ac, as well as H3K27me3, in bulk histones. PARP7 input confirms expected stabilization (2 nd lane from left) and knockdown (3 rd lane from left) of PARP7. (E) IP-western assays showing interactions between PARP7, p300, and C/EBPβ in 3T3-L1 cells. 3T3-L1 cells with Dox-inducible ectopic expression of FLAG-PARP7 were subjected to FLAG IP. The IPs were subjected to western blotting for FLAG, p300, and C/EBPβ. (F and G) Bar graphs showing the enrichment of (F) C/EBPβ or (G) p300 at the promoters of target genes Pparg , Atf3 , and Klf15 , as assayed by ChIP-qPCR, in 3T3-L1 cells differentiated for 24 h using MDI cocktail and subjected to siRNA-mediated depletion of Parp7 . Mean + SEM; n = 3. Asterisks indicate significant differences from control; Student’s t test; ** p < 0.01, *** p < 0.005, **** p < 0.001, and n.s., not significant. (H and I) Bar graphs showing the enrichment of (H) C/EBPβ and (I) H3K27ac at the promoters of target genes Pparg , Atf3 , and Klf15 , as assayed by ChIP-qPCR, in 3T3-L1 cells differentiated for 24 h using MDI cocktail and subjected to treatment with the p300 inhibitor A-485. Mean + SEM; n = 3. Asterisks indicate significant differences from control; Student’s t test; * p < 0.05, ** p < 0.01, and **** p < 0.001.

Article Snippet: Other antibodies used were as follows: PARP7 (Thermo Fisher Scientific, PA5–40774; RRID:AB_2607074), C/EBPβ (Invitrogen, PA5–120052; RRID:AB_2913624, Invitrogen, PA5–86117; RRID:AB_2802916, and Cell Signaling Technology, 3082; RRID:AB_2260365), Phospho-C/EBPβ (Thr235) (Cell Signaling Technology, 3084S; RRID:AB_2260359), PPARγ (81B8) (Cell Signaling Technology, 2443; RRID:AB_823598), cJun (Proteintech, 24909–1-AP; RRID:AB_2860574), cJun (Cell Signaling Technology, 9165; RRID:AB_2130165), DTX2 (ThermoFisher, PA5–109664; RRID:AB_2855075), RNF114 (Proteintech, 14338–1-AP; RRID:AB_3085435), Histone H3K27ac (Active Motif, 39134; RRID:AB_2722569), Histone H2BK5ac (Cell Signaling Technology, 12799; RRID:AB_2636805), Histone H3K27me3 (Cell Signaling Technology, 9733; RRID:AB_2616029), FLAG (Sigma-Aldrich, F3165; RRID:AB_259529), MAR binding reagent (Millipore, MABE1076; RRID:AB_2665469), PAR binding reagent (Millipore, MABE1031; RRID:AB_2665467), Perilipin (Biosynth, 20R-PP004; RRID: AB_3665667), Ubiquitin (E4I2J) (Cell Signaling Technology, 43124; RRID:AB_2799235), K48-linkage specific polyubiquitin (Cell Signaling Technology, 4289; RRID:AB_10557239), p300 (Active Motif, 61404; RRID:AB_2716754), p300 (Santa Cruz, SC-584; RRID:AB_2293429), NAMPT (D1K6D) (Cell Signaling Technology, 61122; RRID:AB_2799602), NAMPT (Proteintech, 11776–1-AP; RRID:AB_2298317), β-tubulin (Abcam, ab6046; RRID:AB_2210370), and β-Actin (8H10D10) (Cell Signaling Technology, 3700; RRID:AB_2242334).

Techniques: Western Blot, Expressing, ChIP-qPCR, Control, Knockdown

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Schematic of the domain organizations of the human cPRC1 complex. Abbreviations of domain names: Chromo, chromodomain; ATHL, AT-hook like; ABM, acidic-patch-binding motif; Pc, Polycomb box; RAWUL, ring finger and WD40 associated ubiquitin-like; PEST, proline, glutamic acid, serine and threonine rich; HD1, homology domain 1; FCS, zinc finger with a characteristic phenylalanine–cysteine–serine sequence motif; SAM, sterile alpha motif. b , In vitro ubiquitination assays of RNF2-BMI1 with unmodified or H2BK120ub-modified nucleosomes, visualized by Coomassie blue staining. The bottom row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 5 (BMI1-NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results c , In vitro ubiquitination assays of RNF20-RNF40 with unmodified or H2AK119ub-modified nucleosomes, analyzed by Western blot. The bottom row represents the ratio of the band intensity of H2B-ub to that of H3, normalized to lane 1 (RNF20-RNF40-NCP H2AK119ub , set to 1.00). The experiment was repeated at least three times with similar results d , Overall structure of cPRC1-E2∼ub-NCP H2BK120ub complex. Cryo-EM density map (upper panel) and atomic model (lower panel) of cPRC1-E2∼ub-NCP H2BK120ub complex are shown from two orthogonal views. The cryo-EM map is segmented according to the components of the cPRC1-E2∼ub-NCP H2BK120ub complex. The color scheme of the cPRC1-E2∼ub-NCP H2BK120ub complex is the same as depicted in (a), and the 147-bp DNA chains are shown in light and dark gray, respectively. Ub, ubiquitin; E2, UbcH5c. e , Detailed view of the recognition interface between H2BK120ub and BMI1. f , In vitro ubiquitination assays of wild-type and mutant cPRC1 complexes with unmodified or H2BK120ub-modified nucleosomes, analyzed by Coomassie blue staining (top) and Western blot (bottom). The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 2 (BMI1 WT -NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Binding Assay, Ubiquitin Proteomics, Sequencing, Sterility, In Vitro, Modification, Staining, Western Blot, Cryo-EM Sample Prep, Mutagenesis

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Schematic of the domain organizations of the human ncPRC1.1 ternary complex. Abbreviations of domain names: NZF, Npl4-type zinc finger; RID, Ring1B-interacting domain; RAWUL, ring finger and WD40 associated ubiquitin-like. b , In vitro ubiquitination assays of ncPRC1.1 and ncPRC1.6 with unmodified or H2BK120ub-modified nucleosomes, analyzed by Coomassie blue staining (top) and Western blot (bottom). The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub, H2A-ub2, and H2A-ub3) to that of H3, normalized to lane 2 (RNF2-PCGF1-NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results. c , Overall structure of human ncPRC1.1-E2∼ub-NCP H2BK120ub complex. Cryo-EM density map (upper panel) and atomic model (lower panel) of ncPRC1.1-E2∼ub-NCP H2BK120ub complex are shown from two orthogonal views. The cryo-EM map is segmented according to the components of ncPRC1.1-E2∼ub-NCP H2BK120ub complex. The color scheme of ncPRC1.1-E2∼ub-NCP H2BK120ub complex is the same as depicted in (a), and the 147-bp DNA chains are shown in light and dark gray, respectively. Ub, ubiquitin; E2, UbcH5c. d , Structural comparison of cPRC1-E2∼ub-NCP H2BK120ub and ncPRC1.1-E2∼ub-NCP H2BK120ub . e , Detailed view of the recognition interface between H2BK120ub and PCGF1.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Ubiquitin Proteomics, In Vitro, Modification, Staining, Western Blot, Cryo-EM Sample Prep, Comparison

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Cryo-EM density maps and atomic models of human ncPRC1.4-NCP H2BK120ub&H2AK119ub complex (left), human ncPRC1.4-NCP H2AK119ub complex (middle), human ncPRC1.1-NCP H2BK120ub&H2AK119ub complex (top right), and human ncPRC1.6-NCP H2BK120ub&H2AK119ub complex (bottom right) in the dyad view of nucleosome. b , Detailed view of the recognition interfaces between RYBP and the nucleosome acidic patch (left), and between RYBP and H2AK119ub (right). c , In vitro ubiquitination assays of wild-type and mutant ncPRC1.1 complexes with unmodified nucleosomes, analyzed by Coomassie blue staining (middle) and Western blot (bottom). The upper panel shows the protein purity of wild-type and mutant ncPRC1.1 complexes loaded at a higher concentration than in the ubiquitination assay. The middle row represents the ratio of the band intensity of H2A-ub (including H2A-ub and H2A-ub2) to that of H3, normalized to lane 2 (RNF2-PCGF1 WT -NCP H2BK120ub , set to 1.00). The experiment was repeated at least three times with similar results. w/o, without RYBP.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Cryo-EM Sample Prep, In Vitro, Ubiquitin Proteomics, Mutagenesis, Staining, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: a , Heatmaps show normalized H2BK120ub, H2AK119ub, Bmi1, H3K4me3, and H3K27me3 signals in WT and Rnf20 KO+Flag-Bmi1 mESCs. Signal differences were calculated by subtracting WT mESC signals from those of Rnf20 -KO+ Flag-Bmi1. Data were plotted across 5 kb windows spanning the TSS to TES of all genes determined by UCSC Genes 2013. Genes were clustered into two groups using K-Means based on WT H2BK120ub and H2AK119ub signals. Heatmaps were sorted by WT H2BK120ub enrichment. TSS, transcription start site. TES, transcription end site. b , Heatmaps of normalized H2BK120ub, H2AK119ub, Bmi1, H3K4me3, and H3K27me3 signals in different gene groups from WT and Rnf20- KO+Flag-Bmi1 mESCs, shown similarly as in . c , Heatmaps (left) show gene expression in different gene groups from WT and Rnf20- KO+Flag-Bmi1 mESCs. Word clouds (medium) illustrate the gene families in each group. GO analysis (right) presents the top four GO terms ranked by P values, with P values calculated using the hypergeometric test. d , Heatmaps (left) display normalized gene expression and a percentage stacked bar plot (right) shows the distribution of average normalized expression in unique_ub, overlap, and unique_me from WT mESCs at differentiation time points of 0, 4, 7, and 14 days. The normalization was applied by rows.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Gene Expression, Expressing

Journal: bioRxiv

Article Title: Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

doi: 10.64898/2026.02.20.706939

Figure Lengend Snippet: Direct interaction between H2BK120ub and specific PCGF subunits promotes PRC1 activity while limiting RYBP association with nucleosomes. Co-enrichment of H2BK120ub and H2AK119ub is observed in the gene bodies of a subset of developmental genes.

Article Snippet: 10 μg of H2BK120ub antibodies (Cell Signaling Technology, Cat. #5546S; Active Motif, Cat. #39623) were added into each sample and rocked at 4 L overnight.

Techniques: Activity Assay