ring1b  (Cell Signaling Technology Inc)

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: Knockdown of RING1A and RING1B promoted tube formation, NO production, and ac-LDL uptake in HUVECs. (A) RT-PCR analysis of RING1A and RING1B expression in control, RING1A KD, and RING1B KD HUVECs. (B) Western blot analysis of indicated proteins in control, RING1A KD, and RING1B KD HUVECs. (C) Quantification of the western blot experiment. (D) Tube formation assay of control, RING1A KD, and RING1B KD HUVECs. (E) Quantification of total vessel length in the tube formation assay. (F) Quantification of total number of junctions in the tube formation assay. (G) NO production assay in control, RING1A KD, and RING1B KD HUVECs. (H) Quantification of the percentage of DAF-FM positive cells in the NO production assay. (I) Quantification of median fluorescence intensity in the NO production assay. (J) Ac-LDL uptake assay in control, RING1A KD, and RING1B KD HUVECs. (K) Quantification of the percentage of ac-LDL positive cells in the ac-LDL uptake assay. (L) Quantification of median fluorescence intensity in the ac-LDL uptake assay. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. RT-PCR, real-time PCR; KD, knockdown; DAF-FM, 4-amino-5-methylamino-2′, 7′-difluorofluorescein; NO, nitric oxide; ac-LDL, acetylated low-density lipoprotein.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Tube Formation Assay, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: Knockdown of RING1A, but not RING1B, inhibited proliferation, cell cycle progression, and migration of HUVECs. (A) BrdU proliferation assay in control, RING1A KD, and RING1B KD HUVECs. (B) Quantification of BrdU positive cells in the proliferation assay. (C) Cell cycle analysis in control, RING1A KD, and RING1B KD HUVECs by PI staining. (D) Quantification of cell cycle phase distribution (G0/G1, S, G2/M). (E) Scratch assay in control, RING1A KD, and RING1B KD HUVECs. (F) Quantification of wound closure rate in the scratch assay. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. KD, knockdown; BrdU, Bromodeoxyuridine; PI, propidium iodide.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Knockdown, Migration, Proliferation Assay, Control, Cell Cycle Assay, Staining, Wound Healing Assay

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: RING1A and RING1B regulate angiogenesis-related gene expression in HUVECs. (A-B) Identification of genes regulated by RING1A or RING1B in HUVECs. Genes that were upregulated in RING1A KD or RING1B KD HUVECs compared to control were labeled as “repressed”, while genes that were downregulated were labeled as “activated”. (C-D) Summary of the GO-BP analysis of RING1A-repressed or RING1B-repressed genes. (E) Venn diagram of RING1A-repressed and RING1B-repressed genes. (F) Top 20 GO-BP terms associated with RING1A and RING1B co-repressed genes. (G) KEGG pathway analysis of RING1A and RING1B co-repressed genes. (H) Top 20 GO-BP terms of RING1A-specific repressed genes. (I) Top 20 GO-BP terms of RING1B-specific repressed genes. (J) Heatmap of expression (z-score-normalized) of RING1A and RING1B co-repressed genes. (K) Heatmap showing the expression of PRC1 components in RING1A KD, RING1B KD, and control HUVECs, normalized to z-scores. (L) GO-BP analysis of genes activated by RING1A (upper) and RING1B (lower), specifically related to cell proliferation. All experiments were independently repeated at least three times. KD, knockdown; GO-BP, Gene Ontology-Biological Process; KEGG, Kyoto Encyclopedia of Genes and Genomes; FC, fold change.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Gene Expression, Control, Labeling, Expressing, Knockdown

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: RING1A and RING1B are enriched at angiogenesis-related genes in HUVECs. (A) RPKM-normalized CUT&Tag signals and heatmap visualization for H2AK119ub-, RING1A-, RING1B-associated genes in HUVECs. (B) Genomic distribution of H2AK119ub, RING1A, and RING1B peaks in HUVECs. (C-E) GO-BP analysis of blood vessel-related pathways for genes enriched with H2AK119ub, RING1A, and RING1B. (F) Heatmap of CUT&Tag signals for H2AK119ub, RING1A, and RING1B target genes, clustered into three groups by enrichment patterns: Cluster 1 (C1) comprises genes with H2AK119ub, RING1A and/or RING1B enrichment; Cluster 2 (C2) comprises genes with RING1A and/or RING1B but not H2AK119ub enrichment; Cluster 3 (C3) comprises genes with H2AK119ub enrichment only. (G & H) Classification of RING1A-repressed or RING1B-repressed genes. “RING1A (RING1B) * & H2AK119ub*” denotes genes repressed by RING1A (RING1B) and enriched with both RING1A (RING1B) and H2AK119ub; “RING1A (RING1B) *” denotes genes repressed by RING1A (RING1B) and enriched with RING1A (RING1B); “H2AK119ub*” denotes genes repressed by RING1A (RING1B) and enriched with H2AK119ub; other classifications indicate genes repressed by RING1A (RING1B) but not enriched with RING1A (RING1B) or H2AK119ub. (I) Log 2 fold change of gene expression for genes denoted in Figure G. (J) Log 2 fold change of gene expression for genes denoted in Figure H. (K) Top 20 biological processes of GO-BP analysis for genes co-repressed and co-enriched by RING1A and RING1B. (L) Signal tracking for angiogenesis-related genes. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. RPKM, reads per kilobase million; GO-BP, Gene Ontology- Biological Process; KD, knockdown; bp, base pair; Kb, kilobase.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Gene Expression, Knockdown

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: BMP4 knockdown abolished the enhanced tube formation induced by RING1A and/or RING1B knockdown in HUVECs. (A) CUT&Tag-PCR analysis of H2AK119ub enrichment at the BMP4 gene locus in control, RING1A KD, RING1B KD, and RING1A/B KD HUVECs. (B) RT-PCR analysis of BMP4 expression in control, RING1A KD, RING1B KD, and RING1A/B KD HUVECs. (C) Western blot analysis of indicated protein. (D) Tube formation assay. (E) Quantification of total vessel length in the tube formation assay. (F) Quantification of total number of junctions in the tube formation assay. (G) Tube formation assay of control, RING1A/B KD HUVECs, supplemented with DMH1 or Noggin. (H) Quantification of total vessel length in the tube formation assay. (I) Quantification of total number of junctions in the tube formation assay. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. RT-PCR, real-time PCR; KD, knockdown.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Knockdown, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Tube Formation Assay, Real-time Polymerase Chain Reaction

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: Knockdown of RING1A or RING1B promotes angiogenesis in both Matrigel plug model and alkaline burn-induced corneal angiogenesis model. (A) Schematic illustration of the Matrigel plug model. (B) RT-PCR analysis of RING1A and RING1B expression in Matrigel plugs mixed with negative control siRNA (NC-siRNA), RING1A-targeting siRNA (siRING1A), and RING1B-targeting siRNA (siRING1B) retrieved from wild-type mice. (C) Images of Matrigel plugs mixed with NC-siRNA, siRING1A, and siRING1B. (D-F) Matrigel plugs were fixed with PFA, sectioned, and subjected to H&E staining (D), CD31 IF staining (E), and quantification of vessel number (F). (G) Schematic illustration of the alkaline burn-induced corneal angiogenesis model. (H) CD31 IF staining of corneas treated with NC-siRNA, siRING1A, and siRING1B. (I) Quantification of the percentage of corneal area occupied by vessels using CD31 IF staining. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. H&E, hematoxylin and eosin; IF, immunofluorescence.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Staining, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Disruption of PRC1 components RING1A and RING1B promotes angiogenesis via relieving BMP4 repression

doi: 10.1016/j.jare.2025.07.025

Figure Lengend Snippet: Inhibition of BMP4 blocks the enhanced angiogenesis induced by RING1A and RING1B knockdown in vivo . (A) Representative images of Matrigel plugs mixed with negative control siRNA (NC-siRNA), RING1A/RING1B-targeting siRNA (siRING1A/B), and siRING1A/B supplemented with DMH1 or Noggin retrieved from wild-type mice. (B) RT-PCR analysis of RING1A and RING1B expression in Matrigel plugs mixed with NC-siRNA, siRING1A/B, and siRING1A/B supplemented with DMH1 or Noggin and retrieved from wild-type mice. (C–E) Matrigel plugs were fixed with PFA, sectioned, and subjected to H&E staining (C), CD31 IF staining (D), and quantification of vessel number in the Matrigel plugs (E). (F) The model of PRC1 regulates angiogenesis in ECs. *p < 0.05, **p < 0.01, ***p < 0.001. The data represent the mean ± SEM. All experiments were independently repeated at least three times. RT-PCR, real-time PCR; H&E, hematoxylin and eosin; IF, immunofluorescence.

Article Snippet: Rabbit monoclonal antibodies to RING1A (13069), RING1B (5694), H2AK119ub (8240), and H3 (4499) were from CST.

Techniques: Inhibition, Knockdown, In Vivo, Negative Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) RING1 and RNF2 variants (top). Reported variants in ClinVar and cancer-related somatic (COSMIC) mutations in RING1 and RNF2 genes (bottom). Metadome plots (middle) represent the level of predicted intolerance for amino acid change in RING1A and RING1B. For COSMIC, only positions of interest are shown as labels. Circle size represents the number of patients reported. (B) ColabFold predictions of RING1A and RING1B variants in altering interaction with PCGF proteins. (C) WBs of dKO-RING1A/B cells expressing HA-tagged WT and mutant RING1A and RING1B. Vinculin and histone H3 served as loading controls. n = 3 independent experimental replicates. (D) Possible mechanisms of deleterious variants that result in a decrease or absence of H2AK119ub. (E) Partial protein sequence alignments of a subset of RING1B homologs. The conserved RING1B-R70 residue corresponds to C. elegans R181 and is indicated by a star. Conserved zinc-coordinating residues, blue ; required for stabilizing the E2 enzyme-E3 ligase interaction in mammals, red ; required for binding to the nucleosome in mammals, green predicted to be important for the RING1B:PCGF4 interaction, magenta 47; and predicted to mediate β sheet interactions, cyan. * indicates identical residues, and : and. indicate residues with strongly and weakly similar physicochemical properties, respectively. The secondary structure of SPAT-3 and H. sapiens RING1B is shown below. (F) WBs of H2AK119ub in the indicated genotypes. The dilution factor is 1:3. The spat-3(mgw26) allele is a full deletion of the spat-3 coding region. Quantification of H2AK119ub and SPAT-3 isoform A is normalized to loading controls (histone H3/actin) and shown relative to the sample indicated by an asterisk. ND, not detectable. (G) WBs in dKO-RING1A/B cells stably expressing HA-RING1B WT or HA-RING1B R70H . Vinculin and histone H2A and H3 served as fractionation controls. n = 3 independent experimental replicates. (H) Normalized H3K27me3 Cut&Run signal (two independent experimental replicates) in cells treated with 1 μM of vehicle (DMSO) or GSK343 for 72 h. See also and .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Expressing, Mutagenesis, Sequencing, Residue, Binding Assay, Stable Transfection, Fractionation

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Strategy to generate Rnf2 WT/R70H ESCs by homologous recombination. (B) DEG from WT and two clones of Rnf2 WT/R70H ESCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) GO of upregulated genes in Rnf2 WT/R70H ESCs. (D) Heatmaps of Ring1b, H3K27me3, and H2AK119ub ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (E) Strategy to generate HA and FLAG-tagged Rnf2 alleles by CRISPR-Cas9 in WT and Rnf2 WT/R70H ESCs. (F) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in WT and Rnf2 WT/R70H ESCs. Signal was generated from two biological replicates from two independent WT and Rnf2 WT/R70H clones. HA and FLAG Cut&Run signals were merged (average of 4 replicates) to avoid potential bias from the HA and FLAG antibodies’ efficiency. (G) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H ESCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as a negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H ESCs. (H) Heatmaps of Cbx7 and Pcgf2, Rybp, Mtf2/Pcl2, and Jarid2 ChIP-seq (average signal of two independent experimental replicates) in WT and clone #1 of Rnf2 WT/R70H ESCs. (I) Genome browser screenshots of ChIP-seq from (H). (J) Mutabind2 scores upon the human RING1B R70H variant vs. full length and lacking their IDR, PCGF1-6 using AlphaFold and ColabFold. (K) Full-length Pcgf2 or lacking the IDR used in (L). (L) Anti-HA IPs followed by WBs against HA, Phc1, and Ring1b in WT and Rnf2 WT/R70H ESCs expressing HA-Pcgf2 WT or HA-Pcgf2 ΔIDR . (M) Model of PRC1/2 recruitment in Rnf2 WT/R70H ESCs. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Homologous Recombination, Clone Assay, ChIP-sequencing, CRISPR, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, Variant Assay, Expressing

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Protocol to generate and differentiate NPCs. (B) Heatmap of DEG from ESCs vs. NPCs vs. differentiated NPCs (log 2 fold > 4, q < 0.01) and between WT and two clones of Rnf2 WT/R70H ESCs, NPCs, and 12-day-old differentiated NPCs (log 2 fold > 2, q < 0.01). n = 2 independent experimental replicates. (C) IFs of neuronal markers in WT and Rnf2 WT/R70H differentiated NPCs. n = 3 independent experimental replicates. Scale bar, 10 μm. (D) WBs of PRC1 subunits from WT ESCs and Pcgf2 KO and Rnf2 WT/R70H with and without Pcgf2 . (E) Pictures of NPCs derived from cells in (D). n = 4 independent experimental replicates. Scale bar, 400 μm. (F) Strategy to generate NPCs expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K in PRC1 CKO cells. (G) WBs of HA and Ring1b in PRC1 CKO cells expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K in the presence and absence of OHT treatment for 48 h. Vinculin was used as a loading control. (H) Pictures of NPCs derived from cells in (G) in a constant presence of OHT treatment. n = 2. Scale bar, 400 μm. (I) WBs of Nanog, Pax6, and H2AK119ub in NPCs derived from PRC1 CKO cells expressing Ring1b WT , Ring1b R70H , or Ring1b I53A/D56K . Vinculin was used as a loading control. (J) UMAP plots of scRNA-seq from 16-day-old WT and two clones of Rnf2 WT/R70H differentiated NPCs. n = 2 independent experimental replicates. (K) Cell type proportions of cells from (E). * p < 0.05. ANOVA test. (L) KEGG pathway of WT and two clones of Rnf2 WT/R70H NPCs. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Clone Assay, Derivative Assay, Expressing, Control

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) Normalized Ring1b WT and Ring1b R70H Cut&Run signals in either WT or Rnf2 WT/R70H NPCs in WT Ring1b peak regions. Two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (B) Normalized H3K27me3 and H2AK119ub Cut&Run signals in either WT or Rnf2 WT/R70H NPCs over all genome. Signal was generated from two biological replicates from two independent clones. Wilcox test. *** p < 0.001. (C) Genome browser screenshots of HA, FLAG, H3K27me3, and H2AK119ub Cut&Run (average signal between replicates) in the cells shown on the left. (D) Anti-FLAG IPs in Rnf2 HA-WT/FLAG-R70H and Rnf2 FLAG-WT/HA-R70H NPCs followed by LC-MS/MS in three independent experimental replicates. Results are normalized to IgG as negative control. Volcano plot shows proteins enriched or weakened in FLAG-Ring1b R70H compared with FLAG-Ring1b WT from Rnf2 WT/R70H NPCs. (E) RNA-seq heatmap of PcG target genes in ESCs that are upregulated in WT NPCs but retained PRC1/2 and are repressed in Rnf2 WT/R70H NPCs. #1 and #2 are two different Rnf2 WT/R70H ESC clones. On the right, GO from each cluster. Deseq2; Wald test (FC > 4), q < 0.05. (F) Simplified genome browser screenshots of Ring1b WT , Ring1b R70H , H3K27me3, and H2AK119ub Cut&Run in WT and Rnf2 WT/R70H NPCs. Ring1b signal in WT NPCs and Ring1b WT and Ring1b R70H signals in Rnf2 WT/R70H NPCs are from merging average signals HA and FLAG Cut&Run two replicates from two clones. (G) Normalized signal of Ring1b WT and Ring1b R70H Cut&Run signals as in (F) around the transcription start site (TSS) of genes from (E). (H) Normalized signal of H3K27me3 and H2AK119ub Cut&Run signals (average from two replicates) as in (F) around the TSS of genes from (E). (I) Normalized ATAC-seq signal (average from two replicates) in WT and Rnf2 WT/R70H ESCs and NPCs around the TSS of genes from (E). See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Clone Assay, Generated, Liquid Chromatography with Mass Spectroscopy, Negative Control, RNA Sequencing

Journal: Molecular cell

Article Title: Unbalanced chromatin binding of Polycomb complexes drives neurodevelopmental disorders

doi: 10.1016/j.molcel.2026.01.023

Figure Lengend Snippet: (A) PCA from ATAC-seq from two independent biological replicates of WT and Rnf2 WT/R70H ESCs and NPCs. (B) Genome browser of ATAC-seq signal (average of two replicates) from WT and Rnf2 WT/R70H ESCs and NPCs. (C) RT-qPCR of pluripotency genes and NPC markers in WT and Rnf2 WT/R70H ESCs and NPCs. n = 3. #1 and #2 represent two clones of Rnf2 WT/R70H ESCs. *** p < 0.005, **** p < 0.001 by ANOVA test. (D) WB of Pax6 in WT and clone #1 of Rnf2 WT/R70H ESCs and NPCs. Vinculin served as a loading control. (E) ATAC-seq peaks reduced in Rnf2 WT/R70H NPCs and HOMER analysis. (F) Normalized expression of genes from (E) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (G) ATAC-seq specific peaks in Rnf2 WT/R70H NPCs and HOMER analysis. (H) Normalized expression of genes from (G) in WT and clones #1 and #2 of Rnf2 WT/R70H NPCs. *** p < 0.001. NS, not significant. Wilcox test. (I) ATAC-seq signal in WT and Rnf2 WT/R70H NPCs at Sox2- or Sox3-occupied sites in WT NPCs. Sox2 and Sox3 ChIP from Bergsland et al. (J) Genome browser of ATAC-seq signal from WT and Rnf2 WT/R70H ESCs and NPCs as well as Ring1b WT and Ring1b R70H Cut&Run signal in WT and Rnf2 WT/R70H NPCs. (K) Normalized expression and GO of genes occupied by Ring1b WT and Ring1b R70H and compacted. *** p < 0.001. NS, not significant. Wilcox test. See also .

Article Snippet: Ring1b (ChIP-seq) , Cell Signaling Technology , Cat# 5694, RRID:AB_10705604.

Techniques: Quantitative RT-PCR, Clone Assay, Control, Expressing