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rabbit polyclonal antibody against ub (Cell Signaling Technology Inc)

Name: rabbit polyclonal antibody against ub
Brand: Cell Signaling Technology Inc
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Cell Signaling Technology Inc rabbit polyclonal antibody against ub
Rabbit Polyclonal Antibody Against Ub, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 1335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal antibody against ub/product/Cell Signaling Technology Inc
Average 98 stars, based on 1335 article reviews

rabbit polyclonal antibody against ub - by Bioz Stars, 2026-03

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(A to C) Clinical scores in the mSOD1 mice treated with anti-RGMa Ab ( n = 12) and control Ab ( n = 12) are shown. ( A ) Anti-RGMa Ab prolonged the survival of mSOD1 mice by a mean of 8.4 days ( P = 0.0065). ( B ) Anti-RGMa Ab significantly mitigated body weight loss ( P = 0.033), shown as the ratio to bodyweight in 10-week-old mice. ( C ) Anti-RGMa Ab in the mSOD1 mice improved their motor function measured by the rotarod test ( P = 0.0099). ( D ) Nissl staining of the ventral horn of the spinal cord in mSOD1 mice is shown. Anti-RGMa Ab administration preserved the number of motor neurons with normal morphology ( n = 5 for the control Ab–treated group and n = 5 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 100 μm. ( E ) Immunohistochemical analysis showed that anti-RGMa Ab treatment reduced the number of ubiquitin-positive neurons in mSOD1 mice ( n = 3 for the control Ab–treated group and n = 7 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 300 μm. ( F ) Immunohistochemical analysis revealed that anti-RGMa Ab administration reduced the accumulation of SOD1 protein ( n = 4 for the control group and n = 6 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 300 μm. Error bars indicate mean ± SEM.

Journal: Science Advances

Article Title: RGMa collapses the neuronal actin barrier against disease-implicated protein and exacerbates ALS

doi: 10.1126/sciadv.adg3193

Figure Lengend Snippet: (A to C) Clinical scores in the mSOD1 mice treated with anti-RGMa Ab ( n = 12) and control Ab ( n = 12) are shown. ( A ) Anti-RGMa Ab prolonged the survival of mSOD1 mice by a mean of 8.4 days ( P = 0.0065). ( B ) Anti-RGMa Ab significantly mitigated body weight loss ( P = 0.033), shown as the ratio to bodyweight in 10-week-old mice. ( C ) Anti-RGMa Ab in the mSOD1 mice improved their motor function measured by the rotarod test ( P = 0.0099). ( D ) Nissl staining of the ventral horn of the spinal cord in mSOD1 mice is shown. Anti-RGMa Ab administration preserved the number of motor neurons with normal morphology ( n = 5 for the control Ab–treated group and n = 5 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 100 μm. ( E ) Immunohistochemical analysis showed that anti-RGMa Ab treatment reduced the number of ubiquitin-positive neurons in mSOD1 mice ( n = 3 for the control Ab–treated group and n = 7 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 300 μm. ( F ) Immunohistochemical analysis revealed that anti-RGMa Ab administration reduced the accumulation of SOD1 protein ( n = 4 for the control group and n = 6 for the anti-RGMa Ab–treated group). Representative images are shown. Scale bar, 300 μm. Error bars indicate mean ± SEM.

Article Snippet: The following primary antibodies were used: mouse NEO1 monoclonal antibody (1:100 dilution, Santa Cruz Biotechnology) (WT, n = 5 and mSOD1 mice, n = 5), rabbit polyclonal antibody against ubiquitin (1:1000 dilution, DAKO) (control antibody group, n = 3 and anti-RGMa antibody group, n = 7), and mouse monoclonal antibody against SOD1 (1:100 dilution, MBL) (control antibody group, n = 4 and anti-RGMa antibody group, n = 6).

Techniques: Staining, Immunohistochemistry

Htt aggregates visualised by staining with the MW8 antibody were present in adjacent sections for IG of R6/2 brains as early as 14 days of age (A, black arrowheads) and increased in number with time (B, C; black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–C’). Cell distribution in this region was visualised with a cresyl violet (CV) stain (A”–C”). Scale bar = 50 µm.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: Htt aggregates visualised by staining with the MW8 antibody were present in adjacent sections for IG of R6/2 brains as early as 14 days of age (A, black arrowheads) and increased in number with time (B, C; black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–C’). Cell distribution in this region was visualised with a cresyl violet (CV) stain (A”–C”). Scale bar = 50 µm.

Article Snippet: Antibodies used were either a rabbit polyclonal antibody raised against poly-ubiquitin (1∶2000; Dako, Denmark) or a mouse monoclonal antibody MW8 raised against Htt (1∶2000; kind gift of Dr. Paul Patterson, Caltech, CA, USA).

Techniques: Staining

Htt aggregates visualised by staining with the MW8 antibody were present in the CTX of R6/2 brains at 16 days in layer V (E, F; black arrowheads) and at 18 days in layer II/III (C, black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–F’). Scale bar = 50 µm.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: Htt aggregates visualised by staining with the MW8 antibody were present in the CTX of R6/2 brains at 16 days in layer V (E, F; black arrowheads) and at 18 days in layer II/III (C, black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–F’). Scale bar = 50 µm.

Techniques: Staining

In R6/2 hippocampus, Htt aggregates visualised by staining with the MW8 antibody could not be seen at 18 days of age but could be found in many neurons at 19 days in the CA1 (B, black arrowheads) and increased in number with time (C, black arrowheads). Cell distribution in this region is shown in a parallel section showed with a cresyl violet stain (A”–C”). In the DG, MW8-positive aggregates were absent up to 24 days, but were present at 26 days (F, black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–F’). Cell distribution in both regions was visualised with a cresyl violet (CV) stain (D”–F”). Scale bar = 50 µm.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: In R6/2 hippocampus, Htt aggregates visualised by staining with the MW8 antibody could not be seen at 18 days of age but could be found in many neurons at 19 days in the CA1 (B, black arrowheads) and increased in number with time (C, black arrowheads). Cell distribution in this region is shown in a parallel section showed with a cresyl violet stain (A”–C”). In the DG, MW8-positive aggregates were absent up to 24 days, but were present at 26 days (F, black arrowheads). Staining with ubiquitin antibody did not reveal the presence of inclusions in any region at these time points (A’–F’). Cell distribution in both regions was visualised with a cresyl violet (CV) stain (D”–F”). Scale bar = 50 µm.

Techniques: Staining

Ubiquitinated Htt aggregates visualised by staining with the ubiquitin antibody (shown here at 14, 19, 22, 24, 26, 29, 41 and 63 days) in IG, CA1, CA3, DG and STR of R6/2 brains. A few inclusions are visible in the IG at 19 days of age. In the hippocampus, aggregates were present at 24 days in CA1, whereas in the CA3 a few aggregates were visible at 41 days of age. In the DG, a few ubiquitin-positive inclusions were present at 41 days. In the striatum, inclusions could be setected at 41 days of age in STR DM and STR VL. The number of aggregates in all the regions increased with time from the day of appearance. Scale bar = 50 µm.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: Ubiquitinated Htt aggregates visualised by staining with the ubiquitin antibody (shown here at 14, 19, 22, 24, 26, 29, 41 and 63 days) in IG, CA1, CA3, DG and STR of R6/2 brains. A few inclusions are visible in the IG at 19 days of age. In the hippocampus, aggregates were present at 24 days in CA1, whereas in the CA3 a few aggregates were visible at 41 days of age. In the DG, a few ubiquitin-positive inclusions were present at 41 days. In the striatum, inclusions could be setected at 41 days of age in STR DM and STR VL. The number of aggregates in all the regions increased with time from the day of appearance. Scale bar = 50 µm.

Techniques: Staining

Regardless of whether or not huntingtin aggregates (visualized by staining with MW8 and ubiquitin antibodies) formed early or late, aggregates that formed in cells with small (DG neurons) and medium-sized (CA1 and STR) soma were found ubiquitinated within 2 to 3 days of initial aggregation. By contrast, there was typically a more than 3 days lag between initial aggregate formation and inclusion ubiquitination in cells with large soma (CA3). Scale bar = 50 µm.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: Regardless of whether or not huntingtin aggregates (visualized by staining with MW8 and ubiquitin antibodies) formed early or late, aggregates that formed in cells with small (DG neurons) and medium-sized (CA1 and STR) soma were found ubiquitinated within 2 to 3 days of initial aggregation. By contrast, there was typically a more than 3 days lag between initial aggregate formation and inclusion ubiquitination in cells with large soma (CA3). Scale bar = 50 µm.

Techniques: Staining

Higher magnification microscopy analysis was used in order to compare MW8 and ubiquitin staining patterns in single R6/2 neurons. In CA1, CA3 and cortex, MW8 labelling could be observed in cells before any aggregates were visible, a phenomenon that was not apparent with ubiquitin staining. The initial morphologies of MW8-positive aggregates were different from ubiquitin-positive inclusions, in that only nucleation centres of the aggregates were ubiquitinated and the MW8-immunolabelled Htt protein that was not localised to the nucleation centre did not appear to be ubiquitinated. In contrast, aggregates with clear nucleation centres were visible by 25 days in the striatum. And at the same time, larger punctate ubiquitin-labelled inclusions were already visible in STR neurones. Ubiquitinated inclusions were much bigger than MW8-positive aggregates and did not change much in size, even though Htt aggregates got bigger with time.

Journal: PLoS ONE

Article Title: Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington’s Disease Mutation

doi: 10.1371/journal.pone.0041450

Figure Lengend Snippet: Higher magnification microscopy analysis was used in order to compare MW8 and ubiquitin staining patterns in single R6/2 neurons. In CA1, CA3 and cortex, MW8 labelling could be observed in cells before any aggregates were visible, a phenomenon that was not apparent with ubiquitin staining. The initial morphologies of MW8-positive aggregates were different from ubiquitin-positive inclusions, in that only nucleation centres of the aggregates were ubiquitinated and the MW8-immunolabelled Htt protein that was not localised to the nucleation centre did not appear to be ubiquitinated. In contrast, aggregates with clear nucleation centres were visible by 25 days in the striatum. And at the same time, larger punctate ubiquitin-labelled inclusions were already visible in STR neurones. Ubiquitinated inclusions were much bigger than MW8-positive aggregates and did not change much in size, even though Htt aggregates got bigger with time.

Techniques: Microscopy, Staining

Pretreatments with LA or/and ALC prevented rotenone-induced accumulation of α-synuclein and ubiquitin Distribution of α-synuclein and ubiquitin were determined by immunofluorescence labelling and confocal microscopy (bar, 10 μm). Images shown are representative of three independent experiments.

Journal: Journal of Cellular and Molecular Medicine

Article Title: Combined R-α–lipoic acid and acetyl-L-carnitine exerts efficient preventative effects in a cellular model of Parkinson’s disease

doi: 10.1111/j.1582-4934.2008.00390.x

Figure Lengend Snippet: Pretreatments with LA or/and ALC prevented rotenone-induced accumulation of α-synuclein and ubiquitin Distribution of α-synuclein and ubiquitin were determined by immunofluorescence labelling and confocal microscopy (bar, 10 μm). Images shown are representative of three independent experiments.

Article Snippet: ADP (high purity, ATP-free) and rotenone were purchased from Sigma (St. Louis, MO, USA); carboxy-H 2- DCFDA, JC-1, MitoTraker, Hoechst 33342, and TRITC-conjugated anti-mouse IgG from Molecular Probes (Eugene, OR, USA); Oxyblot protein oxidation detection kit from Chemicon International Inc. (Temecula, CA, USA); monoclonal anti-8-hydroxyguanine (8-oxo-dG) antibodies from R&D Systems, Inc. (Shanghai, China); BCA protein assay reagent kit from Pierce (Rockford, IL, USA); primary mouse anti-α-synuclein from Lab Vision (Fremont, CA, USA) or from Calbiochem (Darmstadt, Germany); polyclonal rabbit antibody against ubiquitin from DAKO (Carpinteria, CA, USA); monoclonal mouse antibody against complex I subunit (α subcomplex, 9) from Molecular Probes, Invitrogen (Carlsbad, CA, USA); polyclonal rabbit antibody against PGC-1α from Santa Cruz Biotechnology Inc. (San Diego, CA, USA); monoclonal mouse antibody against tubulin from Sigma (St. Louis, MO, USA); IQTM SYBER green supermix reagent from Bio-Rad (Hercules, CA, USA); fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG and Rhodamine (TRITC)-conjugated goat anti-rabbit IgG from Sino-American Biotech Co. (China); Western blotting luminol reagent from Santa Cruz Biotechnology, foetal bovine serum from Hyclone (Logan, UT, USA); penicillin and streptomycin from Invitrogen (Carlsbad, CA, USA); GSH assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); and ALC from Sigma-Tau, Germany.

Techniques: Immunofluorescence, Confocal Microscopy

The ubiquitin-proteasome pathway plays a major role in the degradation of pCH25H during PRRSV infection. (A) PAMs were infected with PRRSV (MOI of 1.0). At various time points (24, 36, 48, and 60 h) postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. (B to F) PAMs were infected with PRRSV (MOI of 1.0) and treated with various inhibitors, including MG132 (10 μM) (B), NH4Cl (10 mM) (C), Z-VAD-FMK (10 μM) (D), CQ (20 μM) (E), or 3-MA (5 mM) (F). At 24, 36, and 48 h postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. ImageJ software was used to analyze the relative levels of pCH25H in comparison with mock-infected cells, and the ratios are displayed as fold changes below the images. DMSO, dimethyl sulfoxide.

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: The ubiquitin-proteasome pathway plays a major role in the degradation of pCH25H during PRRSV infection. (A) PAMs were infected with PRRSV (MOI of 1.0). At various time points (24, 36, 48, and 60 h) postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. (B to F) PAMs were infected with PRRSV (MOI of 1.0) and treated with various inhibitors, including MG132 (10 μM) (B), NH4Cl (10 mM) (C), Z-VAD-FMK (10 μM) (D), CQ (20 μM) (E), or 3-MA (5 mM) (F). At 24, 36, and 48 h postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. ImageJ software was used to analyze the relative levels of pCH25H in comparison with mock-infected cells, and the ratios are displayed as fold changes below the images. DMSO, dimethyl sulfoxide.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Infection, Expressing, Western Blot, Software

PRRSV E protein degrades pCH25H via the ubiquitin-proteasome pathway. (A) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. At 24 h posttransfection, cells were treated for 24 h with MG132 (10 μM), Z-VAD-FMK (10 μM), 3-MA (5 mM), or NH4Cl (10 mM). Cell lysates were harvested and analyzed by Western blotting for analysis of expression of PRRSV E protein and pCH25H. (B) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. Cells were treated with different concentrations of MG132 at 24 h posttransfection. Cell lysates were harvested and analyzed by Western blotting for analysis of pCH25H expression. (C and D) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged E protein and FLAG-tagged pCH25H. Cells were lysed at 48 h posttransfection and immunoprecipitated with anti-FLAG antibody. The whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-β-actin, and anti-ubiquitin (Ub) (C) or anti-K48 (D) antibodies. DMSO, dimethyl sulfoxide.

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: PRRSV E protein degrades pCH25H via the ubiquitin-proteasome pathway. (A) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. At 24 h posttransfection, cells were treated for 24 h with MG132 (10 μM), Z-VAD-FMK (10 μM), 3-MA (5 mM), or NH4Cl (10 mM). Cell lysates were harvested and analyzed by Western blotting for analysis of expression of PRRSV E protein and pCH25H. (B) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. Cells were treated with different concentrations of MG132 at 24 h posttransfection. Cell lysates were harvested and analyzed by Western blotting for analysis of pCH25H expression. (C and D) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged E protein and FLAG-tagged pCH25H. Cells were lysed at 48 h posttransfection and immunoprecipitated with anti-FLAG antibody. The whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-β-actin, and anti-ubiquitin (Ub) (C) or anti-K48 (D) antibodies. DMSO, dimethyl sulfoxide.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Expressing, Western Blot, Immunoprecipitation

The Lys28 site of pCH25H is ubiquitinated by PRRSV E protein. (A) CH25H sequences from different species were aligned (*, lysine). (B) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged wild-type (WT) pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A) and HA-tagged PRRSV E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. The numbers below the images represent the relative levels of pCH25H, compared to that of the corresponding control group, as determined via ImageJ analysis. (C) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged PRRSV E protein and FLAG-tagged wild-type pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A). The cells were lysed at 48 h after transfection and immunoprecipitated with an anti-FLAG antibody. Whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-ubiquitin (Ub), or anti-β-actin antibodies. (D) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged K28A and different amounts of expression vectors encoding HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. (E) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged pCH25H or hCH25H and HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting.

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: The Lys28 site of pCH25H is ubiquitinated by PRRSV E protein. (A) CH25H sequences from different species were aligned (*, lysine). (B) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged wild-type (WT) pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A) and HA-tagged PRRSV E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. The numbers below the images represent the relative levels of pCH25H, compared to that of the corresponding control group, as determined via ImageJ analysis. (C) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged PRRSV E protein and FLAG-tagged wild-type pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A). The cells were lysed at 48 h after transfection and immunoprecipitated with an anti-FLAG antibody. Whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-ubiquitin (Ub), or anti-β-actin antibodies. (D) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged K28A and different amounts of expression vectors encoding HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. (E) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged pCH25H or hCH25H and HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Expressing, Western Blot, Transfection, Immunoprecipitation

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Ubiquitin Proteomics, Infection, Expressing, Western Blot, Software, Comparison

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Transfection, Immunoprecipitation, Ubiquitin Proteomics

FAT10 stimulates both the inhibitory and deubiquitylating activity of OTUB1 toward TRAF3. A, lysates of HEK293 cells transiently expressing Flag-TRAF3, HA-ubiquitin, Y2-OTUB1, and myc-FAT10 were subjected to immunoprecipitation (IP) using anti-HA–agarose followed by Western blot (IB) analysis using directly labeled anti-Flag or anti-HA antibodies or primary antibodies against myc or the C-terminal part of GFP. β-Actin was used as a loading control. B, HEK293 cells were transiently transfected with the indicated expression plasmids, and lysates were subjected to anti-HA immunoprecipitation and Western blot analysis as described in A. C, after transient transfection with the indicated expression plasmids, HEK293 cells were subjected to procedures and treatments as described in A. One representative experiment of three experiments with similar outcomes is shown.

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-like modifier FAT10 stimulates the activity of deubiquitylating enzyme OTUB1

doi: 10.1074/jbc.RA118.005406

Figure Lengend Snippet: FAT10 stimulates both the inhibitory and deubiquitylating activity of OTUB1 toward TRAF3. A, lysates of HEK293 cells transiently expressing Flag-TRAF3, HA-ubiquitin, Y2-OTUB1, and myc-FAT10 were subjected to immunoprecipitation (IP) using anti-HA–agarose followed by Western blot (IB) analysis using directly labeled anti-Flag or anti-HA antibodies or primary antibodies against myc or the C-terminal part of GFP. β-Actin was used as a loading control. B, HEK293 cells were transiently transfected with the indicated expression plasmids, and lysates were subjected to anti-HA immunoprecipitation and Western blot analysis as described in A. C, after transient transfection with the indicated expression plasmids, HEK293 cells were subjected to procedures and treatments as described in A. One representative experiment of three experiments with similar outcomes is shown.

Article Snippet: For analysis of ubiquitin conjugates, a polyclonal rabbit antibody against ubiquitin was applied (Z0458, DakoCytomation, Hamburg, Germany).

Techniques: Activity Assay, Expressing, Immunoprecipitation, Western Blot, Labeling, Transfection

FAT10 and USE1 enhance the cleavage activity of OTUB1 in vitro. A, recombinant Lys-48 or Lys-63 DiUb (5 μm) was incubated with tagless OTUB1 (5 μm) for the indicated time periods at 37 °C, and reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. Proteins were separated on 4–12% NuPAGE bis-Tris gels with subsequent colloidal Coomassie staining. B, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with His-tagged or tagless OTUB1 WT or C91S (all 10 μm) for the indicated time periods at 30 °C. C, silver-stained gel of recombinant Lys-48–linked DiUb (5 μm) incubated for the indicated time periods with tagless OTUB1 (5 μm) and tagless FAT10, UbcH5B-His, and His-USE1 (all 10 μm) at 37 °C. The reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. D, densitometric analysis of cleaved Lys-48–linked DiUb by OTUB1 in the presence of FAT10, USE1, and UbcH5B. The DiUb protein amount at time point 0 was set to 100%. E, enlarged focus on the first 10 min of the densitometric analysis in D. F, FRET-based analysis for the cleavage of internally quenched Lys-48 DiUb (400 nm) by OTUB1 WT or C91S (both 30 nm) in the absence or presence of FAT10, USE1, and UbcH5B (all 5 μm). One representative experiment of three experiments with similar outcomes is shown.

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-like modifier FAT10 stimulates the activity of deubiquitylating enzyme OTUB1

doi: 10.1074/jbc.RA118.005406

Figure Lengend Snippet: FAT10 and USE1 enhance the cleavage activity of OTUB1 in vitro. A, recombinant Lys-48 or Lys-63 DiUb (5 μm) was incubated with tagless OTUB1 (5 μm) for the indicated time periods at 37 °C, and reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. Proteins were separated on 4–12% NuPAGE bis-Tris gels with subsequent colloidal Coomassie staining. B, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with His-tagged or tagless OTUB1 WT or C91S (all 10 μm) for the indicated time periods at 30 °C. C, silver-stained gel of recombinant Lys-48–linked DiUb (5 μm) incubated for the indicated time periods with tagless OTUB1 (5 μm) and tagless FAT10, UbcH5B-His, and His-USE1 (all 10 μm) at 37 °C. The reactions were stopped by the addition of gel sample buffer supplemented with 4% 2-mercaptoethanol. D, densitometric analysis of cleaved Lys-48–linked DiUb by OTUB1 in the presence of FAT10, USE1, and UbcH5B. The DiUb protein amount at time point 0 was set to 100%. E, enlarged focus on the first 10 min of the densitometric analysis in D. F, FRET-based analysis for the cleavage of internally quenched Lys-48 DiUb (400 nm) by OTUB1 WT or C91S (both 30 nm) in the absence or presence of FAT10, USE1, and UbcH5B (all 5 μm). One representative experiment of three experiments with similar outcomes is shown.

Article Snippet: For analysis of ubiquitin conjugates, a polyclonal rabbit antibody against ubiquitin was applied (Z0458, DakoCytomation, Hamburg, Germany).

Techniques: Activity Assay, In Vitro, Recombinant, Incubation, Staining, Fluorescence

The noncovalent interaction of FAT10 with OTUB1 stimulates its DUB activity in cellulo and in vitro. A and B, HEK293 cells were transiently transfected with HA-ubiquitin, Flag-OTUB1 and Flag-FAT10 WT, or Flag-FAT10 AV. Cell lysates were subjected to anti-HA immunoprecipitation (IP) and Western blot (IB) analysis using anti-HA and anti-Flag peroxidase–conjugated antibodies. C, lysates of HEK293 cells transiently expressing HA-ubiquitin WT, HA-ubiquitin K48only, HA-ubiquitin K63only (remaining Lys residues were mutated to Arg), Flag-OTUB1, and Flag-FAT10 were subjected to immunoprecipitation using anti-HA–coupled agarose and Western blot analysis. β-Actin was used as the loading control in A–C. D, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with OTUB1 (10 μm) in the absence or presence of FAT10, UbcH5B, and USE1 (5 μm) for the indicated time periods at 30 °C. One representative experiment of three experiments with similar outcomes is shown.

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-like modifier FAT10 stimulates the activity of deubiquitylating enzyme OTUB1

doi: 10.1074/jbc.RA118.005406

Figure Lengend Snippet: The noncovalent interaction of FAT10 with OTUB1 stimulates its DUB activity in cellulo and in vitro. A and B, HEK293 cells were transiently transfected with HA-ubiquitin, Flag-OTUB1 and Flag-FAT10 WT, or Flag-FAT10 AV. Cell lysates were subjected to anti-HA immunoprecipitation (IP) and Western blot (IB) analysis using anti-HA and anti-Flag peroxidase–conjugated antibodies. C, lysates of HEK293 cells transiently expressing HA-ubiquitin WT, HA-ubiquitin K48only, HA-ubiquitin K63only (remaining Lys residues were mutated to Arg), Flag-OTUB1, and Flag-FAT10 were subjected to immunoprecipitation using anti-HA–coupled agarose and Western blot analysis. β-Actin was used as the loading control in A–C. D, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with OTUB1 (10 μm) in the absence or presence of FAT10, UbcH5B, and USE1 (5 μm) for the indicated time periods at 30 °C. One representative experiment of three experiments with similar outcomes is shown.

Article Snippet: For analysis of ubiquitin conjugates, a polyclonal rabbit antibody against ubiquitin was applied (Z0458, DakoCytomation, Hamburg, Germany).

Techniques: Activity Assay, In Vitro, Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Incubation

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