anti kdm1a  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Proteomic Screening Identifies HNRNPA2B1 as an Epigenetic Repressor of Epstein-Barr Virus Reactivation

doi: 10.64898/2026.02.27.708571

Figure Lengend Snippet: ( A ) WB analysis of histone modifications in Akata (EBV+) cells following HNRNPA2B1 depletion and IgG-crosslinking-induced lytic reactivation. Whole-cell lysates were collected at the indicated time points and probed for H3K4Me3, H3K27Ac, total H3, H2AK5Ac, and total H2A. β-actin serves as a loading control. ( B ) ChIP-qPCR analysis of H3K4Me3 enrichment at EBV ZTAp and RTAp in control (sg-NC) and HNRNPA2B1-depleted (sg2-A2B1) Akata (EBV+) cells. Data are shown as relative to 2% of input. Data are presented as mean ± SD from three biological replicates. ***p<0.001. ( C ) Plasmids expressing Halo-HA-KDMs and Halo-V5-HNRNPA2B1 were co-transfected into HEK-293T cells as indicated. Cell lysates containing the indicated tagged proteins were IP-ed with anti-HA antibody-conjugated beads and analyzed by WB with anti-V5 and anti-HA antibodies. Co-IP analysis showing stronger interaction between HNRNPA2B1 and KDM1A. β-actin serves as a loading control. ( D-F ) WB analysis of EBV lytic protein ZTA and KDM1A ( D ), KDM5A ( E ), or KDM5D ( F ) expression in KDM1A-, KDM5A-, or KDM5D-depleted SNU-719 cells following lytic induction by TPA treatment. β-actin serves as a loading control. ( G ) Co-IP analysis validating the interaction between HNRNPA2B1 and KDM1A. HEK-293T cells were transfected with Halo-HA-KDM1A and Halo-V5-HNRNPA2B1 as indicated. Cell lysates were treated with or without benzonase, followed by IP using anti-HA antibody-conjugated beads. IP and input samples were analyzed by WB with anti-V5 and anti-HA antibodies. β-actin serves as a loading control. ( H ) Proximity ligation assay (PLA) demonstrating the interaction between HNRNPA2B1 and KDM1A in situ . Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. Subsequently, the cells were incubated with either PBS control or a combination of mouse anti-KDM1A and rabbit anti-HNRNPA2B1 antibodies. Probes were then added for ligation and amplification. Cell nuclei were visualized using Nikon AXR after staining with 4′,6-diamidino-2-phenylindole (DAPI). The interaction between HNRNPA2B1 and KDM1A in situ was indicated by red dot representing PLA signals. Scale bars, 10 µm.

Article Snippet: DNA-protein complex were immunoprecipitated with anti-RNA polymerase II (Cat. #05-623-25UG, Milipore Sigma), anti-HNRNPA2B1 (Cat. # 67445-1-Ig, Proteintech), anti-H3K4Me3 (Cat. # 9751S, Cell Signaling Technology), anti-KDM1A (Cat. #2139S, Cell Signaling Technology), rabbit IgG control (Cat. #2729, Cell Signaling Technology), and mouse IgG control (Cat. #sc-2025, Santa Cruz).

Techniques: Control, ChIP-qPCR, Expressing, Transfection, Co-Immunoprecipitation Assay, Proximity Ligation Assay, In Situ, Saline, Incubation, Ligation, Amplification, Staining

Journal: bioRxiv

Article Title: Proteomic Screening Identifies HNRNPA2B1 as an Epigenetic Repressor of Epstein-Barr Virus Reactivation

doi: 10.64898/2026.02.27.708571

Figure Lengend Snippet: ( A ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata (EBV+) cells transduced with non-targeting control (sg-NC) or HNRNPA2B1-targeting sgRNA (sg2-A2B1). Anti-KDM1A was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. *p<0.05; **p<0.01. ( B ) Akata (EBV+) cells carrying control sg-NC or HNRNPA2B1-targeting sgRNA (sg2-A2B1) were treated with anti-human IgG for the indicated times. Protein levels of HNRNPA2B1 and KDM1A were analyzed by WB. β-actin serves as a loading control. ( C ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata-BX1 cells expressing control sg-NC or HNRNPA2B1-targeting sgRNA (sg2-A2B1). Anti-KDM1A was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. ***p<0.001. ( D ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata-BX1 sgA2B1 cells reconstituted with vector control or HNRNPA2B1 (pLenti-A2B1). Anti-KDM1A antibody was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. *p<0.05; ***p<0.001.

Article Snippet: DNA-protein complex were immunoprecipitated with anti-RNA polymerase II (Cat. #05-623-25UG, Milipore Sigma), anti-HNRNPA2B1 (Cat. # 67445-1-Ig, Proteintech), anti-H3K4Me3 (Cat. # 9751S, Cell Signaling Technology), anti-KDM1A (Cat. #2139S, Cell Signaling Technology), rabbit IgG control (Cat. #2729, Cell Signaling Technology), and mouse IgG control (Cat. #sc-2025, Santa Cruz).

Techniques: ChIP-qPCR, Transduction, Control, Negative Control, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Proteomic Screening Identifies HNRNPA2B1 as an Epigenetic Repressor of Epstein-Barr Virus Reactivation

doi: 10.64898/2026.02.27.708571

Figure Lengend Snippet: ( A ) WB analysis of histone modifications in Akata (EBV+) cells following HNRNPA2B1 depletion and IgG-crosslinking-induced lytic reactivation. Whole-cell lysates were collected at the indicated time points and probed for H3K4Me3, H3K27Ac, total H3, H2AK5Ac, and total H2A. β-actin serves as a loading control. ( B ) ChIP-qPCR analysis of H3K4Me3 enrichment at EBV ZTAp and RTAp in control (sg-NC) and HNRNPA2B1-depleted (sg2-A2B1) Akata (EBV+) cells. Data are shown as relative to 2% of input. Data are presented as mean ± SD from three biological replicates. ***p<0.001. ( C ) Plasmids expressing Halo-HA-KDMs and Halo-V5-HNRNPA2B1 were co-transfected into HEK-293T cells as indicated. Cell lysates containing the indicated tagged proteins were IP-ed with anti-HA antibody-conjugated beads and analyzed by WB with anti-V5 and anti-HA antibodies. Co-IP analysis showing stronger interaction between HNRNPA2B1 and KDM1A. β-actin serves as a loading control. ( D-F ) WB analysis of EBV lytic protein ZTA and KDM1A ( D ), KDM5A ( E ), or KDM5D ( F ) expression in KDM1A-, KDM5A-, or KDM5D-depleted SNU-719 cells following lytic induction by TPA treatment. β-actin serves as a loading control. ( G ) Co-IP analysis validating the interaction between HNRNPA2B1 and KDM1A. HEK-293T cells were transfected with Halo-HA-KDM1A and Halo-V5-HNRNPA2B1 as indicated. Cell lysates were treated with or without benzonase, followed by IP using anti-HA antibody-conjugated beads. IP and input samples were analyzed by WB with anti-V5 and anti-HA antibodies. β-actin serves as a loading control. ( H ) Proximity ligation assay (PLA) demonstrating the interaction between HNRNPA2B1 and KDM1A in situ . Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. Subsequently, the cells were incubated with either PBS control or a combination of mouse anti-KDM1A and rabbit anti-HNRNPA2B1 antibodies. Probes were then added for ligation and amplification. Cell nuclei were visualized using Nikon AXR after staining with 4′,6-diamidino-2-phenylindole (DAPI). The interaction between HNRNPA2B1 and KDM1A in situ was indicated by red dot representing PLA signals. Scale bars, 10 µm.

Article Snippet: Briefly, Akata (EBV+) cells were blocked with 3% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4□°C with either PBS control or a mixture of rabbit anti-HNRNPA2B1 (Cat. #14813-1-AP, Proteintech) and mouse anti-KDM1A (Cat. #67037-1-Ig, Proteintech) antibodies (1:50 in 3% BSA).

Techniques: Control, ChIP-qPCR, Expressing, Transfection, Co-Immunoprecipitation Assay, Proximity Ligation Assay, In Situ, Saline, Incubation, Ligation, Amplification, Staining

Journal: bioRxiv

Article Title: Proteomic Screening Identifies HNRNPA2B1 as an Epigenetic Repressor of Epstein-Barr Virus Reactivation

doi: 10.64898/2026.02.27.708571

Figure Lengend Snippet: ( A ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata (EBV+) cells transduced with non-targeting control (sg-NC) or HNRNPA2B1-targeting sgRNA (sg2-A2B1). Anti-KDM1A was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. *p<0.05; **p<0.01. ( B ) Akata (EBV+) cells carrying control sg-NC or HNRNPA2B1-targeting sgRNA (sg2-A2B1) were treated with anti-human IgG for the indicated times. Protein levels of HNRNPA2B1 and KDM1A were analyzed by WB. β-actin serves as a loading control. ( C ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata-BX1 cells expressing control sg-NC or HNRNPA2B1-targeting sgRNA (sg2-A2B1). Anti-KDM1A was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. ***p<0.001. ( D ) ChIP-qPCR analysis of KDM1A occupancy at ZTAp and RTAp in Akata-BX1 sgA2B1 cells reconstituted with vector control or HNRNPA2B1 (pLenti-A2B1). Anti-KDM1A antibody was used for KDM1A ChIP and IgG served as a negative control. Data are normalized to 2% input and shown as mean ± SD from three biological replicates. *p<0.05; ***p<0.001.

Article Snippet: Briefly, Akata (EBV+) cells were blocked with 3% BSA in PBS for 1 hour at room temperature, then incubated overnight at 4□°C with either PBS control or a mixture of rabbit anti-HNRNPA2B1 (Cat. #14813-1-AP, Proteintech) and mouse anti-KDM1A (Cat. #67037-1-Ig, Proteintech) antibodies (1:50 in 3% BSA).

Techniques: ChIP-qPCR, Transduction, Control, Negative Control, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome

doi: 10.1101/2025.03.06.641037

Figure Lengend Snippet: A. Complete interactome of ADNP in hNSCs derived from ChIP-MS. Nodes are colored based on their function or complex association. red: GTF2I/KDM1A complex, orange: ChAP complex, pink: EHMT1/2 complex, blue: POGZ interactors, light blue: TFIIIC complex, dark green: ATRX complex. Known physical interactions excluding ADNP were derived from Stringdb and reported. B. ChIP-seq input signal for KDM1A and GTF2I in WT hNSCs. Each row represents a 5Kb window centered on KDM1A and GTF2I peak summits, respectively. C. Distribution of peaks shared by ADNP-KDM1A-GTF2I (AKG) across different genomic features with respect to protein-coding genes. D. Heatmap of KDM1A ChIP-seq signal in ADNP WT and ADNP KO hNSCs. Each row represents a 5Kb window centered on peak summits, sorted by the KDM1A ChIP signal. Peaks are divided into ADNP-bound (top), and ADNP-free (bottom) based on effective ADNP binding in the WT line. E. Percentage of DEGs in hNSCs bound by individual AKG components or the whole complex. Significance value for each TF binding is reported. F. Heatmap representing the percentage of peaks found between pairs of ENCODE TFs, located within +2500bp and −2500bp from the TSS of protein-coding genes. A red square highlights a cluster made by the top 10,000 peaks of ADNP (“ADNP Top”) and peaks of KDM1A and GTF2I (in bold). All ADNP peaks are indicated as “ADNP All” in the bottom row of the heatmap. G. Scatterplot representing the percentage of ADNP peaks contained in ENCODE TF binding sites of H9 hESC (y-axis) and percentage of ENCODE peaks contained in CTL-FLAG iPSCs ADNP binding sites. Dots are colored by log2(%X*%Y).

Article Snippet: For KDM1A and GTF2I immunoprecipitation, 250 µL of F2V5-ADNP hNSC nuclear extract with 38 units of Benzonase (Novagen) and 1x CEF protease inhibitor (Roche) were rotated with 782 ng KDM1A antibody (Cell Signaling, 2139S) or 300 ng of GTF2I antibody (Bethyl, A301-330A) for 2 hours at 4°C in no-stick microcentrifuge tubes (Alpha Laboratories).

Techniques: Derivative Assay, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Disruption of ADNP-KDM1A-GTF2I complex drives neural differentiation imbalance in Helsmoortel-Van der Aa syndrome

doi: 10.1101/2025.03.06.641037

Figure Lengend Snippet: A. Schematic representation of the interactome of ADNP in hNSCs. Interactors are grouped by known complex identity and coloured accordingly. See also Figure S3A and Table S3 . B. Western blot analysis of immunoprecipitations with KDM1A antibody or an IgG control from hNSC nuclear extract. KDM1A (upper panel) and ADNP (lower panel) are detected by KDM1A antibody and FLAG antibody, respectively. Molecular weight (MW) markers are indicated. C. Western blot analysis of immunoprecipitations with GTF2I antibody or an IgG control from NSC nuclear extract. GTF2I (upper panel) and ADNP (lower panel) are detected by GTF2I antibody and FLAG antibody, respectively. MW markers are indicated. D. Heatmap of ADNP, KDM1A and GTF2I ChIP-seq enrichments in hNSCs. Each row represents a 5kb window centered on ADNP peak summits, sorted by the TF ChIP-seq signal. ChIP-seq input signals for each TF are shown in Figure S3B . ChIP-seq reads have been normalized by library size (1x) as reads per million coverage (RPGC). Normalized signal legend with range is reported below each heatmap. E. UpSet plot indicating the number of significant peaks shared between ADNP-KDM1A-GTF2I (AKG). For the peaks shared by all three TFs, the top DNA binding motif is shown. F. Barplot reporting the enrichment of the ADNP motif in the top 10K peaks identified for each protein (ADNP, GTF2I, KDM1A and KDM1A upon ADNP KO respectively). Motif Enrichment was calculated with Gimmemotif v0.18 upon de novo motif enrichment analysis. G. Barplot showing the portion (%) of DEGs bound by AKG (purple) or ADNP (orange) that lose KDM1A at their promoters upon ADNP KO. H. Scatter plot of H3K4me3 signal in WT (x-axis) vs KO (y-axis). Both axes report hNSC ChIP-seq reads, normalized on library size (RPGC, see Methods) and scaled on the respective input data.

Article Snippet: For KDM1A and GTF2I immunoprecipitation, 250 µL of F2V5-ADNP hNSC nuclear extract with 38 units of Benzonase (Novagen) and 1x CEF protease inhibitor (Roche) were rotated with 782 ng KDM1A antibody (Cell Signaling, 2139S) or 300 ng of GTF2I antibody (Bethyl, A301-330A) for 2 hours at 4°C in no-stick microcentrifuge tubes (Alpha Laboratories).

Techniques: Western Blot, Control, Molecular Weight, ChIP-sequencing, Binding Assay