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anti ha tag antibody chip grade ab9110  (Bethyl)


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    Bethyl anti ha tag antibody chip grade ab9110
    Anti Ha Tag Antibody Chip Grade Ab9110, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/a300+358a/bio_rxiv__64898__2026__03__17__712509-257-18-26?v=Bethyl
    Average 93 stars, based on 59 article reviews
    anti ha tag antibody chip grade ab9110 - by Bioz Stars, 2026-07
    93/100 stars

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    Bethyl anti ha tag antibody chip grade ab9110
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    Screening of epigenetic bifunctionals <t>yields</t> <t>CBP/p300</t> ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.
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    Screening of epigenetic bifunctionals <t>yields</t> <t>CBP/p300</t> ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.
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    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 decreases chromatin accessibility at p300 and EWS/FLI targets. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. Chromatin accessibility changes at selected EWS/FLI targets are shown ( NR0B1, CCND1, FCGRT, GSTM4 ). B) HOMER motif analysis of consensus ATAC-seq peaks lost by aTAG-2 (lost in aTAG-2 vs. DMSO and aTAG-2 vs. binders comparisons). C) Enrichr gene ontology analysis on unique genes corresponding to consensus aTAG-2 lost ATAC-seq peaks (ChEA gene set database). D) Genomic location of consensus aTAG-2 lost ATAC-seq peaks or all ATAC-seq peaks (see Methods ) as reference. E) Overlap of FKBP F36V -HA-EWS/FLI peaks with p300 peaks (MACS2-based peak calling) in DMSO-treated FKBP F36V -HA-EWS/FLI EWS502 cells. P -value calculated by hypergeometric test. ATAC-seq signal heatmaps for HA/p300 co-bound peaks and p300-only bound peaks are shown. HA-only bound peaks are shown in .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques:

    aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 induces targeted chromatin accessibility changes. FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 5 nM for 16 hours and profiled by ATAC-seq. A) GGAA repeat length in consensus aTAG-2 lost peaks that are EWS/FLI bound (Riggi et al. EWS/FLI bound enhancers ) (left) or EWS/FLI-bound ATAC stable peaks (right). B) Enrichr gene ontology analysis on unique genes corresponding to top: consensus aTAG-2 lost ATAC-seq peaks, bottom: consensus aTAG-2 gained ATAC-seq peaks (GEO gene set database). C) Venn diagram of unique genes corresponding to consensus aTAG-2 lost/gained ATAC-seq peaks and EWS/FLI target gene sets from Kinsey et al. (upregulated or downregulated). P -values calculated by hypergeometric test. D) ATAC-seq heatmap across HA-bound, p300-unbound peaks are shown (determined using venn diagram in ).

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques:

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control

    Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: Screening of epigenetic bifunctionals yields CBP/p300 ligand GNE-781 as the strongest transactivator. A) Chemical architecture of a representative aTAG showing the FKBP F36V ligand (AP1867), the activator ligand (purple) and a linker; lower table summarizes the 13 aTAG molecules synthesized, the chromatin effector they recruit, reporter EC 50 values (note: for biphasic curves such as aTAG-5/aTAG-6, this represents the interpolated dose with maximal activation) and maximal activation (fold-change vs. untreated at a single dose) from 10-point dose titration in an IRF1 reporter system (48h treatment); U2OS reporter system stably expressing 1) a plasmid containing 4 repeats of the IRF1 consensus sequence upstream of firefly luciferase, 2) a plasmid containing doxycycline-inducible IRF1-FKBP F36V -mCherry in pLIX403. B) Scatter-plot of maximal activation versus potency for all compounds after 48h treatment. C) Chemical structures of linker series tested for GNE-781 bifunctionals. D) Extended dose curves of AP1867-GNE781 bifunctionals or binders alone (GNE-781, mAP1867-CONHEt) in IRF1 reporter system (normalized FLuc luminescence at 48 hours). E) Extended dose curves of aTAG-2 with binder competition in IRF1 reporter system (normalized FLuc luminescence at 48 hours). F-G) Normalized NLuc luminescence from NanoBiT experiment in 293T cells, titrated with aTAG-2 ± competitor or binder alone for 24 hours. NanoBiT constructs used were FKBP F36V -LgBiT and SmBiT fused to the C-terminus of either the CBP (F) or p300 (G) bromodomain.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: Synthesized, Activation Assay, Titration, Stable Transfection, Expressing, Plasmid Preparation, Sequencing, Luciferase, Construct

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites and degrades FKBP F36V -EWS/FLI in a E1/proteasome-dependent manner. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours were profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at Riggi et al. EWS/FLI bound sites are shown. B) Overlap between unique genes with consensus CBP ChIP-seq peaks gained by aTAG-2 (gained both vs. DMSO and vs. binders), and consensus p300 ChIP-seq peaks lost by aTAG-2. P -value calculated by hypergeometric test. C-D) Enrichr gene ontology analysis (C) and Homer motif analysis (D) of dual CBP-gained and p300-lost peaks/unique genes. E) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of inhibitors (1 μM, except MG-132 at 10 μM; TAK-243: UBA1 inhibitor, TAK-981: SUMOylation inhibitor, MG-132: proteasome inhibitor, lanes are from the same blot & exposure). F) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Total ubiquitin loading control is shown in . G) Model for aTAG-2 mechanism of action, where it can induce transactivation, partial target degradation, and inhibit p300/CBP through a RIPTAC-like mechanism depending on context.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Western Blot, Ubiquitin Proteomics, Control

    aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 replaces p300 with CBP at EWS/FLI bound sites. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by CBP, p300, and HA ChIP-seq. P300 and CBP occupancy heatmaps at consensus aTAG-2 HA-EWS/FLI lost regions are shown (N=551, csaw-based differential binding). B) IGV tracks showing changes in ATAC, CBP, p300, and HA occupancy at the best characterized EWS/FLI target gene NR0B1 .

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay

    aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Journal: bioRxiv

    Article Title: An epigenetic bifunctional that toggles between transactivation and repression

    doi: 10.64898/2026.03.17.712509

    Figure Lengend Snippet: aTAG-2 degrades FKBP F36V -HA-tagged EWS/FLI on chromatin. A) FKBP F36V -HA-EWS/FLI EWS502 cells treated with DMSO, aTAG-2, or binders (mAP1867-CONHEt + GNE-781) at 10 nM for 16 hours and profiled by HA ChIP-seq. The number of HA peaks in MACS2-based peak calling is plotted between the conditions. B) Gene set enrichment analysis (Enrichr, GEO database) on unique genes corresponding to aTAG-2 HA-EWS/FLI consensus lost regions (N=551, csaw-based differential binding). C) Western blot for total ubiquitin (FK2) in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 1 hour following TUBE2 (ubiquitin) pulldown, in the presence of MG-132 (10 μM). Loading control for . D) Western blot for HA-tagged EWS/FLI in FKBP F36V -HA-EWS/FLI EWS502 cells treated with aTAG-2 at 10 nM for 3 hours in the presence of p300/CBP histone acetyl transferase inhibitor A-485 (10 μM). E) Western blot for HA-tagged EWS/FLI and TRIM8 in FKBP F36V -HA-EWS/FLI EWS502 cells ± dox-inducible TRIM8 sgRNAs treated with aTAG-2 at 10 nM for 16 hours.

    Article Snippet: Dynabeads Protein A (50 μL) were washed 3 times with TBS-T then pre-incubated with 10 μg of antibody (anti-HA tag antibody - ChIP Grade (ab9110), P300 (Bethyl, A300-358A), CBP (Bethyl, A300-362A)) at 4C for 3 hours in TBS-T (500 μL).

    Techniques: ChIP-sequencing, Binding Assay, Western Blot, Ubiquitin Proteomics, Control