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: IgG (CUT&RUN) , EpiCypher , Cat# 13-0042, RRID:AB_2923178.

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: IgG (CUT&RUN) , EpiCypher , Cat# 13-0042, RRID:AB_2923178.

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) 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: IgG (CUT&RUN) , EpiCypher , Cat# 13-0042, RRID:AB_2923178.

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: IgG (CUT&RUN) , EpiCypher , Cat# 13-0042, RRID:AB_2923178.

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

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 1. Exogenous PCSK9 can induce degradation of the LDLR in the absence of APLP2. HepG2 (A) and Huh7 (B) cells were transfected with a control non-target siRNA (Ctrl) or 3 different siRNAs targeting APLP2. Cells wereincubatedovernightwithserum-freeconditionedmedialackingorcon- taining 1 g/ml of PCSK9-V5. HepG2 and Huh7 cell lysates were then sub- jected to Western blotting using LDLR, APLP2, and -actin antibodies. LDLR and APLP2 signals were normalized to that of -actin. C, the input HEK293 conditioned medium was analyzed using mAb-V5 to detect PCSK9-V5. D, duplicate samples of Huh7 cells matching those in panel B were analyzed by FACS to assess the cell surface LDLR levels. Values were normalized to that of the first lane (control non-target siRNA in the absence of PCSK9). Error bars represent S.E. *, p 0.05 (Student’s t test). The data shown here are represen- tative of two to three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 4. Sortilin and APLP2 are novel cellular targets of PCSK9. A, overexpressed PCSK9 induces sortilin and APLP2 degradation in HEK293 cells. Triplicate Western blot analyses revealing that both sortilin-Myc and APLP2-V5 expression levels in HEK293 cells were reduced by 90 and 40%, respectively, upon transfection with a PCSK9 plasmid, as compared with a control empty pIRES vector (V). Quantification of sortilin and APLP2 band intensities were normalized against those of -actin. B, HEK293 cells transfected with a cDNA coding for an empty vector control (pIRES; V) or individually with human sortilin or APLP2 tagged at the C terminus with a Myc or V5 epitope, respectively, or together in the absence or presence of a cDNA coding for untagged PCSK9. The following day the cells were washed and then pulsed for 4 h with [35S]Met Cys in the presence or absence of 5 mM NH4Cl. The cells were then extracted and their lysates immunoprecipitated (IP) with a mAb-V5 or mAb-Myc or a polyclonal antibody for PCSK9. The precipitates were separated on an 8% SDS-PAGE. The dried gel was then autoradiographed. Notice the co-precipitation of sortilin and APLP2 in the presence of NH4Cl. These data are representative of at least three independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Immunoprecipitation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 5. Sortilin, APLP2, and soluble APLP2 are degraded by both PCSK9 and ER-localized PCSK9-KDEL isoforms. HEK293 cells were transfected with indicated DNA amounts of vectors encoding a control protein 7B2, sortilin (no tag), APLP2-V5, soluble APLP2-V5 (sAPLP2-V5), PCSK9-V5, or PCSK9-V5-KDEL, as indicated. After 48 h, lysates and media were analyzed by Western blotting for the indicated proteins. The data show that overexpressed PCSK9 or PCSK9-KDEL induces degradation of sortilin (A), APLP2 (B), and sAPLP2 (C) in HEK293 cells. Quantification of sortilin and APLP2 band intensities were normalized against those of -actin or GAPDH. These data are representative of two independent experiments.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Transfection, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 6. Co-expression of sortilin, APLP2, or both with PCSK9 has no major effect on LDLR degradation. Huh7 cells were transfected with a total of 3 g using 1 g of each vector encoding for either a control protein 7B2 (), sortilin (), APLP2 (), or PCSK9 (), as indicated. After 48 h, lysates were analyzed by Western blotting for expression of the LDLR, sortilin-Myc, APLP2- V5, intracellular pro- and mature-PCSK9-V5, and -actin. Media were ana- lyzed for secreted endogenous and overexpressed PCSK9-V5 using a rabbit polyclonal human PCSK9 antibody. Quantification of LDLR expression was normalized against that of -actin. These data are representative of at least 3 different experiments showing similar results.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Amyloid Precursor-like Protein 2 and Sortilin Do Not Regulate the PCSK9 Convertase-mediated Low Density Lipoprotein Receptor Degradation but Interact with Each Other

doi: 10.1074/jbc.m115.647180

Figure Lengend Snippet: FIGURE 7. Sortilin binds APLP2. A, schematic diagram of sortilin and APLP2 fused to the G. princeps luciferase half-domains, Gluc-1 and Gluc-2, respectively. B, heat map generated by G. princeps luciferase complementation assay showing the interaction profile of 36 protein pairs. Normalized luminescence ratio ranging from strong to null interactions is displayed on a light blue to black scale. C, validation of sortilin-Myc and APLP2-V5 interaction was confirmed by co-expression in HEK293 cells and immunoprecipitation with mAb-Myc followed by Western blotting (WB) using a mAb-Myc or mAb-V5.

Article Snippet: Mouse APLP2 was detected using a rabbit polyclonal antibody kindly provided by Dr. G. Thinakaran (University of Chicago), whereas human APLP2 was detected with either mAb-V5 or a rabbit polyclonal antibody (Novus Biologicals).

Techniques: Luciferase, Generated, Biomarker Discovery, Expressing, Immunoprecipitation, Western Blot

Journal: The Journal of biological chemistry

Article Title: Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.

doi: 10.1074/jbc.M512786200

Figure Lengend Snippet: FIGURE 5. Anti-polyubiquitin staining of affinity-purified PHF-Tau. Following SDS- PAGE separation of MC1 affinity-purified PHF-Tau on a 10% polyacrylamide gel, Western blot was performed with an anti-polyubiquitin antibody. Immunoreactivity was visual- ized by enhanced chemiluminescence (left panel). Secondary antibody (goat anti-mouse IgG-horseradish peroxidase) control for nonspecific immunoreactivity is also depicted (right panel).

Article Snippet: Blots were incubated with a monoclonal mouse anti-polyubiquitin antibody (Novus Biologicals) 1:500 overnight at 4 °C.

Techniques: Staining, Affinity Purification, SDS Page, Western Blot, Control

Journal: The Journal of biological chemistry

Article Title: Alzheimer disease-specific conformation of hyperphosphorylated paired helical filament-Tau is polyubiquitinated through Lys-48, Lys-11, and Lys-6 ubiquitin conjugation.

doi: 10.1074/jbc.M512786200

Figure Lengend Snippet: FIGURE 6. Relative quantification of Lys-6, Lys-11, and Lys-48 polyubiquitin linkages by SRM. Polyubiquitin linkages at Lys-6 (A), Lys-11 (B), and Lys-48 (C) of ubiquitin (ubiquitinated and unmodified peptides) were quantified by specific precursor-to-product ion transition monitoring (Table 4). D shows chromatograms for all three ubiquitinated peptides on the same scale for visual comparison of the relative peak areas.

Article Snippet: Blots were incubated with a monoclonal mouse anti-polyubiquitin antibody (Novus Biologicals) 1:500 overnight at 4 °C.

Techniques: Quantitative Proteomics, Ubiquitin Proteomics, Comparison

Journal: The Journal of infectious diseases

Article Title: Hepatitis C Virus nonstructural 5A protein inhibits lipopolysaccharide-mediated apoptosis of hepatocytes by decreasing expression of Toll-like receptor 4.

doi: 10.1093/infdis/jir381

Figure Lengend Snippet: Figure 1. Hepatitis Cvirus (HCV) nonstructural protein 5 A (NS5A) protects hepatocytes from lipopolysaccharide (LPS)–induced cell death. A, B, HepG2 control (A) and HepG2-NS5A (B) hepatocytes were cultured for 24 hours with ligands of Toll-like receptor (TLR) 1/TLR2 (Pam3CSK4), TLR3 (poly[I:C]), TLR4 (LPS), TLR5 (flagellin), TLR6/TLR2 (macrophage-activating lipopeptide 2), TLR7 (Imiquimod [R-837]), TLR9 (type B CpG oligonucleotide), and control oligonucleotide, as indicated in Materials and Methods. Cells were washed and stained with crystal violet, and experiments were performed 3 times. PBS, phosphate-buffered saline. C, HCV NS5A protects hepatocytes from LPS-induced apoptosis. HepG2 control and HepG2-NS5A hepatocytes were cultured for 24 hours with LPS (5 lg/mL). Cell apoptosis was quantified using the APOPercentage Apoptosis Assay. Data are expressed as means 6 standard deviations of triplicate determinations from 1 experiment represen- tative of 3 independent experiments.

Article Snippet: 3HCL; 100 lg/mL), TLR3 (poly[I:C]; 50 lg/mL), TLR4 (LPS from Escherichia coli; 5 lg/mL), TLR5 (purified flagellin; 100 lg/mL), TLR6/TLR2 (macrophage-activating lipopeptide 2; 100 lg/mL), TLR7 (Imiquimod [R-837]; 2.5 lg/mL), and TLR9 (type B CpG ODN; 0.5 lg/mL) (all purchased from Imgenex).

Techniques: Control, Cell Culture, Staining, Saline, Apoptosis Assay