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hrd1  (ABclonal Biotechnology)
94
ABclonal Biotechnology hrd1
HERPUD1 recruits <t>HRD1</t> to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.
Hrd1, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/SYVN1+Rabbit+pAb/pmc13386943-181-8-20
Average 94 stars, based on 1 article reviews
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hrd1  (Proteintech)
94
Proteintech hrd1
HERPUD1 recruits <t>HRD1</t> to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.
Hrd1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/HRD1+Fusion+Protein/pm41862645-194-35-36
Average 94 stars, based on 1 article reviews
hrd1 - by Bioz Stars, 2026-10
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rabbit anti hrd1  (Proteintech)
94
Proteintech rabbit anti hrd1
HERPUD1 recruits <t>HRD1</t> to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.
Rabbit Anti Hrd1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/HRD1+Fusion+Protein/pm41807387-571-40-44
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rabbit anti hrd1 - by Bioz Stars, 2026-10
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anti hrd1  (Cell Signaling Technology Inc)
86
Cell Signaling Technology Inc anti hrd1
HERPUD1 recruits <t>HRD1</t> to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.
Anti Hrd1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/pm41593065-246-9-12
Average 86 stars, based on 1 article reviews
anti hrd1 - by Bioz Stars, 2026-10
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anti hrd1  (Proteintech)
94
Proteintech anti hrd1
HERPUD1 recruits <t>HRD1</t> to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.
Anti Hrd1, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/HRD1+Fusion+Protein/pm41593065-241-35-36
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anti hrd1 - by Bioz Stars, 2026-10
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rabbit α syvn hrd1  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc rabbit α syvn hrd1
a. Illustration of the ER membrane topology of the model ERAD substrate H2a-BAP-SV5 together with cytosolic BirA and the luminal sec-BirA, highlighting three proposed retrotranslocation mechanisms that allow cytosolic BAP biotinylation. b. Western blot of HEK 293 cells expressing H2a-BAP alone or together with BirA or sec-BirA after incubation with Biotin (100µM, 30 min). Biotinylated H2a-BAP was detected with StAv-HRP and total H2a-BAP with α-SV5. c, d. Immunofluorescence of U2OS cells expressing H2a-BAP without BirA untreated or upon bortezomib (Bz, 2.5µM, 3 hours) treatment, stained with α-SV5 and StAv-Cy2 to visualize total and background biotin signal; yellow dashed ROIs mark the ERQC and white boxes indicate zoomed regions, shown on the right. e, f. Localization of total and biotinylated H2a-BAP in U2OS cells expressing the H2a-BAP – BirA system under basal and Bz-treated conditions. g. Quantification of total and biotinylated H2a-BAP in the ERQC relative to whole-cell signal (mean ± SD, n=28; unpaired t-test with Welch’s correction). h-k. Colocalization of biotinylated H2a-BAP with endogenous ER/ERQC markers, Sec61β and calreticulin (CRT). l-n . Colocalization of biotinylated H2a-BAP with ERAD components, endogenous <t>HRD1</t> and transfected HERP-FLAG. Scale bars= 10 µm. o. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with total-H2a-BAP, Sec61β, CRT, HRD1, BiP, and GalT (BiP and GalT from Suppl. Fig. 1) in untreated and Bz-treated cells (unpaired T-test with Welch’s correction).
Rabbit α Syvn Hrd1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hrd1/SYVN1+Rabbit+mAb/bio_rxiv__64898__2026__02__04__703732-215-21-23
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rabbit α syvn hrd1 - by Bioz Stars, 2026-10
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Image Search Results


HERPUD1 recruits HRD1 to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.

Journal: Journal of Virology

Article Title: HERPUD1 suppresses porcine epidemic diarrhea virus replication by recruiting HRD1 to degrade viral ORF3 protein

doi: 10.1128/jvi.00626-26

Figure Lengend Snippet: HERPUD1 recruits HRD1 to regulate the ubiquitin-mediated degradation of ORF3. ( A ) HEK293T cells were transfected with HA-HRD1 and Flag-ORF3, or with Flag-ORF3 alone. All cells were treated with 20 μM MG132 for 12 h, and then analyzed by co-IP and Western blot using the indicated antibodies. ( B ) HRD1 colocalizes with ORF3 in the cytoplasm. ST cells were co-transfected with Flag-ORF3 and HA- HRD1 for 12 or 24 h. Cells were immunostained with mouse anti-ORF3 and rabbit anti-HA antibodies, followed by DAPI staining, and imaged by confocal microscopy. Scale bars, 20 µm. Colocalization analysis was performed on the images within the white dashed boxes using IMAGEJ. ( C–F ) HERPUD1 promotes the interaction between ORF3 and HRD1. The cells were analyzed for protein abundance by Western blot. HEK293T cells were transfected for 24 h with HA-HRD1, Flag-ORF3, and GFP-HERPUD1, followed by co-IP with anti-Flag binding beads. Precipitated proteins were analyzed by Western blot ( C ). HEK293T cells were transfected for 24 h with Flag-HRD1, GFP-ORF3, and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-Flag beads. Precipitated proteins were analyzed by Western blot ( D ). HEK293T cells were transfected for 24 h with Flag-ORF3 and HA-HERPUD1, followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( E ). HEK293T cells were transfected for 24 h with Flag-ORF3 and siRNA (HERPUD1 siRNA or negative-control siRNA), followed by co-IP with anti-HRD1 binding beads. Precipitated proteins were analyzed by Western blot ( F ). ( G ) HRD1 is required for HERPUD1-mediated degradation of ORF3. HEK293T cells were co-transfected with Flag-ORF3, HA-HERPUD1, and either Flag-HRD1 or HRD1-specific siRNA (with non-targeting siRNA as a control). ORF3 protein levels were quantified by Western blot. ( H ) HRD1 modulates K63-linked ubiquitination of ORF3. HEK293T cells were co-transfected with Flag-ORF3, GFP-HRD1, and plasmids encoding HA-tagged WT ubiquitin (HA-Ub-K11O, HA-Ub-K48O, HA-Ub-K63O). Cell lysates were harvested at 24 h post-transfection and subjected to immunoprecipitation with an anti-Flag antibody, followed by Western blot. The experiment was performed three independent times. Relative band intensities were quantified using ImageJ software.

Article Snippet: Antibodies targeting DDDDK (AE092), HA (AE105), Myc (AE070), HRD1 (A2605), Phospho-PERK (60004-1), ATF6 (A0202), and EGFP (AE078) were obtained from ABclonal.

Techniques: Ubiquitin Proteomics, Transfection, Co-Immunoprecipitation Assay, Western Blot, Staining, Confocal Microscopy, Quantitative Proteomics, Binding Assay, Negative Control, Control, Immunoprecipitation, Software

HERPUD1 suppresses PEDV replication by recruiting HRD1 to degrade viral ORF3 protein. PEDV ORF3 protein upregulated expression of HERPUD1 via the ATF6 pathway. Subsequently, HERPUD1 recruits the E3 ubiquitin ligase HRD1 to facilitate ORF3 ubiquitination and subsequent degradation via ERAD, thereby alleviating ORF3-induced ER stress and suppressing PEDV replication.

Journal: Journal of Virology

Article Title: HERPUD1 suppresses porcine epidemic diarrhea virus replication by recruiting HRD1 to degrade viral ORF3 protein

doi: 10.1128/jvi.00626-26

Figure Lengend Snippet: HERPUD1 suppresses PEDV replication by recruiting HRD1 to degrade viral ORF3 protein. PEDV ORF3 protein upregulated expression of HERPUD1 via the ATF6 pathway. Subsequently, HERPUD1 recruits the E3 ubiquitin ligase HRD1 to facilitate ORF3 ubiquitination and subsequent degradation via ERAD, thereby alleviating ORF3-induced ER stress and suppressing PEDV replication.

Article Snippet: Antibodies targeting DDDDK (AE092), HA (AE105), Myc (AE070), HRD1 (A2605), Phospho-PERK (60004-1), ATF6 (A0202), and EGFP (AE078) were obtained from ABclonal.

Techniques: Expressing, Ubiquitin Proteomics

a. Illustration of the ER membrane topology of the model ERAD substrate H2a-BAP-SV5 together with cytosolic BirA and the luminal sec-BirA, highlighting three proposed retrotranslocation mechanisms that allow cytosolic BAP biotinylation. b. Western blot of HEK 293 cells expressing H2a-BAP alone or together with BirA or sec-BirA after incubation with Biotin (100µM, 30 min). Biotinylated H2a-BAP was detected with StAv-HRP and total H2a-BAP with α-SV5. c, d. Immunofluorescence of U2OS cells expressing H2a-BAP without BirA untreated or upon bortezomib (Bz, 2.5µM, 3 hours) treatment, stained with α-SV5 and StAv-Cy2 to visualize total and background biotin signal; yellow dashed ROIs mark the ERQC and white boxes indicate zoomed regions, shown on the right. e, f. Localization of total and biotinylated H2a-BAP in U2OS cells expressing the H2a-BAP – BirA system under basal and Bz-treated conditions. g. Quantification of total and biotinylated H2a-BAP in the ERQC relative to whole-cell signal (mean ± SD, n=28; unpaired t-test with Welch’s correction). h-k. Colocalization of biotinylated H2a-BAP with endogenous ER/ERQC markers, Sec61β and calreticulin (CRT). l-n . Colocalization of biotinylated H2a-BAP with ERAD components, endogenous HRD1 and transfected HERP-FLAG. Scale bars= 10 µm. o. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with total-H2a-BAP, Sec61β, CRT, HRD1, BiP, and GalT (BiP and GalT from Suppl. Fig. 1) in untreated and Bz-treated cells (unpaired T-test with Welch’s correction).

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a. Illustration of the ER membrane topology of the model ERAD substrate H2a-BAP-SV5 together with cytosolic BirA and the luminal sec-BirA, highlighting three proposed retrotranslocation mechanisms that allow cytosolic BAP biotinylation. b. Western blot of HEK 293 cells expressing H2a-BAP alone or together with BirA or sec-BirA after incubation with Biotin (100µM, 30 min). Biotinylated H2a-BAP was detected with StAv-HRP and total H2a-BAP with α-SV5. c, d. Immunofluorescence of U2OS cells expressing H2a-BAP without BirA untreated or upon bortezomib (Bz, 2.5µM, 3 hours) treatment, stained with α-SV5 and StAv-Cy2 to visualize total and background biotin signal; yellow dashed ROIs mark the ERQC and white boxes indicate zoomed regions, shown on the right. e, f. Localization of total and biotinylated H2a-BAP in U2OS cells expressing the H2a-BAP – BirA system under basal and Bz-treated conditions. g. Quantification of total and biotinylated H2a-BAP in the ERQC relative to whole-cell signal (mean ± SD, n=28; unpaired t-test with Welch’s correction). h-k. Colocalization of biotinylated H2a-BAP with endogenous ER/ERQC markers, Sec61β and calreticulin (CRT). l-n . Colocalization of biotinylated H2a-BAP with ERAD components, endogenous HRD1 and transfected HERP-FLAG. Scale bars= 10 µm. o. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with total-H2a-BAP, Sec61β, CRT, HRD1, BiP, and GalT (BiP and GalT from Suppl. Fig. 1) in untreated and Bz-treated cells (unpaired T-test with Welch’s correction).

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Membrane, Western Blot, Expressing, Incubation, Immunofluorescence, Staining, Transfection

a. HEK 293 cells expressing H2a-BAP - BirA were left untreated or treated for 3 hours with Bz (2.5µM), NB-DNJ (200µM) or Kifunensine (Kif, 100µM), alone or with Bz, and lysates were analyzed for total (α-SV5) and biotinylated H2a-BAP (StAv-HRP) or subjected to StAv-agarose pulldown to detect precipitated (α-SV5) and ubiquitylated H2a-BAP (α-Ubiquitin). Deglycosylated species are indicated by red arrows. b, c. Biotinylated H2a-BAP and polyubiquitylated biotinylated H2a-BAP were quantified and normalized to total or StAv-precipitated H2a-BAP, respectively. d. HEK 293 cells co-expressing H2a-BAP - BirA with dominant-negative HRD1 (HRD1-DN) were analyzed under basal or Bz-treated conditions using the same biochemical readouts as in (a). e, f. Quantification of biotinylated and polyubiquitylated H2a-BAP (from d). Statistics were performed using ordinary two-way ANOVA with Tukey’s multiple-comparisons test. g-i. U2OS cells co-expressing H2a-BAP - BirA with HRD1-WT or HRD1-DN, with or without Bz, were stained for total and biotinylated H2a-BAP and HRD1/HRD1-DN (α-Myc) to visualize the impact of HRD1 activity and proteasome inhibition on H2a-BAP localization. j. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with HRD1-DN under untreated and Bz-treated cells (unpaired T-test with Welch’s correction). Scale bars= 10 µm.

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a. HEK 293 cells expressing H2a-BAP - BirA were left untreated or treated for 3 hours with Bz (2.5µM), NB-DNJ (200µM) or Kifunensine (Kif, 100µM), alone or with Bz, and lysates were analyzed for total (α-SV5) and biotinylated H2a-BAP (StAv-HRP) or subjected to StAv-agarose pulldown to detect precipitated (α-SV5) and ubiquitylated H2a-BAP (α-Ubiquitin). Deglycosylated species are indicated by red arrows. b, c. Biotinylated H2a-BAP and polyubiquitylated biotinylated H2a-BAP were quantified and normalized to total or StAv-precipitated H2a-BAP, respectively. d. HEK 293 cells co-expressing H2a-BAP - BirA with dominant-negative HRD1 (HRD1-DN) were analyzed under basal or Bz-treated conditions using the same biochemical readouts as in (a). e, f. Quantification of biotinylated and polyubiquitylated H2a-BAP (from d). Statistics were performed using ordinary two-way ANOVA with Tukey’s multiple-comparisons test. g-i. U2OS cells co-expressing H2a-BAP - BirA with HRD1-WT or HRD1-DN, with or without Bz, were stained for total and biotinylated H2a-BAP and HRD1/HRD1-DN (α-Myc) to visualize the impact of HRD1 activity and proteasome inhibition on H2a-BAP localization. j. Pearson’s R values (Coloc2) for colocalization of biotinylated H2a-BAP with HRD1-DN under untreated and Bz-treated cells (unpaired T-test with Welch’s correction). Scale bars= 10 µm.

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Ubiquitin Proteomics, Dominant Negative Mutation, Staining, Activity Assay, Inhibition

a, b. H2a-BAP – BirA expressing HEK 293 cells, left untreated (a) or treated with Bz (2.5 µM, 3 hours) (b), were homogenized and post-nuclear supernatants were layered on iodixanol step gradients (10, 16, 22, 28 and 34%) and centrifuged at 24,000 r.p.m. for 16 hours. 1 mL fractions were collected from the top and analyzed by Western blot with StAv-HRP to detect biotinylated H2a-BAP and with α-SV5 (or α-Myc) for total H2a-BAP, together with organelle markers α-CNX, α-HRD1, α-Sec61β, α-20S, α-RPL26 and α-Cab45; the deglycosylated H2a-BAP species is indicated by a red arrow. c . H2a-Myc lacking the BAP tag was co-expressed with BirA as a negative control for biotinylation. d. For each protein, the signal in every fraction was expressed as a percentage of total and plotted versus fraction number; one representative experiment of three is shown. Yellow shading marks fractions enriched in membrane-bound organelles, with fractions 1–2 containing soluble proteins and low-density organelles (Golgi), and green shading marks the ERQC peak. e. HEK 293 cells expressing H2a-BAP - BirA were treated with the p97 inhibitor CB5083 (2.5 µM, 3 hours), alone or with Bz (2.5µM, 3 hours), and analyzed by Western blot. f. Glycosylated and deglycosylated biotinylated H2a-BAP species (from (e)) were quantified, normalized to total H2a-BAP (α-SV5) and expressed relative to untreated cells (mean of 5 experiments ± SD; ordinary two-way ANOVA with Tukey’s multiple-comparisons test).

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: a, b. H2a-BAP – BirA expressing HEK 293 cells, left untreated (a) or treated with Bz (2.5 µM, 3 hours) (b), were homogenized and post-nuclear supernatants were layered on iodixanol step gradients (10, 16, 22, 28 and 34%) and centrifuged at 24,000 r.p.m. for 16 hours. 1 mL fractions were collected from the top and analyzed by Western blot with StAv-HRP to detect biotinylated H2a-BAP and with α-SV5 (or α-Myc) for total H2a-BAP, together with organelle markers α-CNX, α-HRD1, α-Sec61β, α-20S, α-RPL26 and α-Cab45; the deglycosylated H2a-BAP species is indicated by a red arrow. c . H2a-Myc lacking the BAP tag was co-expressed with BirA as a negative control for biotinylation. d. For each protein, the signal in every fraction was expressed as a percentage of total and plotted versus fraction number; one representative experiment of three is shown. Yellow shading marks fractions enriched in membrane-bound organelles, with fractions 1–2 containing soluble proteins and low-density organelles (Golgi), and green shading marks the ERQC peak. e. HEK 293 cells expressing H2a-BAP - BirA were treated with the p97 inhibitor CB5083 (2.5 µM, 3 hours), alone or with Bz (2.5µM, 3 hours), and analyzed by Western blot. f. Glycosylated and deglycosylated biotinylated H2a-BAP species (from (e)) were quantified, normalized to total H2a-BAP (α-SV5) and expressed relative to untreated cells (mean of 5 experiments ± SD; ordinary two-way ANOVA with Tukey’s multiple-comparisons test).

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Western Blot, Negative Control, Membrane

H2a-BAP – BirA expressing HEK 293 cells were processed as in but using iodixanol gradients optimized to achieve higher resolution of low and medium density fractions (10%, 13%, 16%, 20% and 24%) under three microsome conditions: untreated microsomes (M), SDS/Triton X-100/NaDOC lysis (S) or Na₂CO₃ extraction (C). a. 1-mL fractions were collected and immunoblotted for biotinylated H2a-BAP (StAv-HRP), deglycosylated species are indicated by red arrows), total H2a-BAP (α-SV5) and calnexin (CNX). b. Parallel blots were probed for endogenous HRD1 and cytosolic or cytosol-facing membrane proteins (α-20S, α-RPL26 and α-eIF2α). c-f. Signals for biotinylated, total, glycosylated and deglycosylated H2a-BAP were quantified for each condition (M, S, C), expressed as percentage of total signal and compared between untreated and Bz-treated cells; one representative experiment of three is shown. g. Scheme of alkaline carbonate extraction release of peripherally membrane-associated proteins from microsomes while retaining integral ER membrane proteins.

Journal: bioRxiv

Article Title: ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

doi: 10.64898/2026.02.04.703732

Figure Lengend Snippet: H2a-BAP – BirA expressing HEK 293 cells were processed as in but using iodixanol gradients optimized to achieve higher resolution of low and medium density fractions (10%, 13%, 16%, 20% and 24%) under three microsome conditions: untreated microsomes (M), SDS/Triton X-100/NaDOC lysis (S) or Na₂CO₃ extraction (C). a. 1-mL fractions were collected and immunoblotted for biotinylated H2a-BAP (StAv-HRP), deglycosylated species are indicated by red arrows), total H2a-BAP (α-SV5) and calnexin (CNX). b. Parallel blots were probed for endogenous HRD1 and cytosolic or cytosol-facing membrane proteins (α-20S, α-RPL26 and α-eIF2α). c-f. Signals for biotinylated, total, glycosylated and deglycosylated H2a-BAP were quantified for each condition (M, S, C), expressed as percentage of total signal and compared between untreated and Bz-treated cells; one representative experiment of three is shown. g. Scheme of alkaline carbonate extraction release of peripherally membrane-associated proteins from microsomes while retaining integral ER membrane proteins.

Article Snippet: Streptavidin-HRP (Jackson ImmunoResearch-016-030-084; 1:5000-7500; WB), Rabbit-α-SV5 (CellSignalling-D3H8Q; 1:2000 (WB), 1:750 (IF), 1:250 (ExM)), Mouse-α-SV5 (GeneScript-A01724-100; 1:1000 (WB), 1:750 (IF), 1:250 (ExM)), Rabbit-α-SYVN (Hrd1) (CellSignaling-14773; 1:1000 (WB), 1:100 (IF)), Rabbit-α-H2a (against N-terminus of H2a as described in ) Rabbit-α-Calnexin (Abcam-92573; 1:5000 (WB)), Rabbit-α-RPL26 (CellSignaling-5400; 1:1000 (WB)), Mouse-α-eIF2α (MBL-AT6031; 1:1000 (WB)), Rabbit-α-C9 (20S) (Abcam-ab118902; 1:1000 (WB)), Rabbit-α-Calnexin (SigmaAldrich - C4731; 1:1000 (WB), 1:500 (IF)), Rabbit-α-Calreticulin (CellSignaling-D3E6; 1:400 (IF)).

Techniques: Expressing, Lysis, Extraction, Membrane