rabbit anti cul4a nature Search Results


resource source identifier antibodies rabbit anti cul4a bethyl laboratories cat  (Bethyl)
93
Bethyl resource source identifier antibodies rabbit anti cul4a bethyl laboratories cat
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anti cul4a 2699s antibody  (Cell Signaling Technology Inc)
95
Cell Signaling Technology Inc anti cul4a 2699s antibody
Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, <t>Cul4A</t> FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.
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rabbit anti cul4a nature  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc rabbit anti cul4a nature
Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, <t>Cul4A</t> FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.
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rabbit anti-cul4a  (Santa Cruz Biotechnology)
90
Santa Cruz Biotechnology rabbit anti-cul4a
Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, <t>Cul4A</t> FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.
Rabbit Anti Cul4a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti cul4a  (Santa Cruz Biotechnology)
96
Santa Cruz Biotechnology rabbit anti cul4a
Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, <t>Cul4A</t> FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.
Rabbit Anti Cul4a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-cul4a (rabbit polyclonal, #2699)  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc anti-cul4a (rabbit polyclonal, #2699)
Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, <t>Cul4A</t> FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.
Anti Cul4a (Rabbit Polyclonal, #2699), supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti cul4a  (Proteintech)
93
Proteintech anti cul4a
E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, <t> Cul4a, </t> and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.
Anti Cul4a, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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rabbit anti-cul4a  (Rockland Immunochemicals)
90
Rockland Immunochemicals rabbit anti-cul4a
E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, <t> Cul4a, </t> and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.
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rabbit anti-cul4a  (GeneTex)
90
GeneTex rabbit anti-cul4a
E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, <t> Cul4a, </t> and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.
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polyclonal rabbit anti-cullin-4a  (Millipore)
90
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A GSCs transduced with control or RNAi targeting CDH1, <t>CUL4A,</t> UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.
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rabbit anti cul4a  (Proteintech)
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Proteintech rabbit anti cul4a
A GSCs transduced with control or RNAi targeting CDH1, <t>CUL4A,</t> UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.
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anti cul4a  (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology anti cul4a
A GSCs transduced with control or RNAi targeting CDH1, <t>CUL4A,</t> UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.
Anti Cul4a, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, Cul4A FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.

Journal: The Journal of Biological Chemistry

Article Title: Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun

doi: 10.1074/jbc.RA118.002246

Figure Lengend Snippet: Quantitative proteomics and biochemical analysis reveal c-Jun as a protein down-regulated by CRBN. A, c-Jun tryptic peptides identified by the quantitative proteomics analysis. The peptide sequence, modification, MH+, heavy to light (H/L) ratio, and Sequest cross correlation score Xcorr were included in the peptide list. B, MS/MS spectrum of a representative tryptic peptide identified from c-Jun. The peptide sequence, MH+, m/z, and Δmass were depicted in the annotated MS/MS spectrum. C, representative MS spectrum of a tryptic peptide used for quantification of the c-Jun protein level. The peptide sequence, MH+, m/z, Δmass, and heavy to light ratio were depicted. The m/z value for the isotope peaks from the light and heavy samples were labeled. D, CRBN overexpression reduces the c-Jun protein level. HEK293T cells were transfected with the pcDNA3.1 or HA-CRBN plasmid using PEI transfection reagent for 48 h. Cell lysates were analyzed by Western blotting (WB) with the indicated antibodies. The means and standard deviations were depicted, and the p value was calculated using Student's t test with data from three biological replicates. **, p < 0.01. E, siRNA knockdown CRBN increases the c-Jun protein level. HEK293T cells were transfected with siNC and siCRBN using Lipofectamine 2000 for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; **, p < 0.01. F, Cul4A FL, but not the deletion mutant Cul4A(Δ), decreases the c-Jun protein level. HEK293T cells were transfected with Cul4A FL or the Cul4A(Δ) plasmid using PEI transfection reagent for 48 h. Samples and data were analyzed as described in D. *, p < 0.05; ns, not significant. G, the c-Jun protein level is increased in brain tissue of Crbn knockout mice. Brain lysates obtained from WT and Crbn knockout mice were immunoblotted with the indicated antibodies. Experiments were carried out with three biological replicates, and quantification was performed as described in D. **, p < 0.01.

Article Snippet: Antibodies used in this work were from the following companies: anti-HA (sc-7392) and anti-ubiquitin (Ub, sc-8017) antibodies from Santa Cruz Biotechnology (USA); anti-Cul4A (2699S) antibody from Cell Signaling Technology (USA); anti-c-Jun (CPA1634), anti-COX-2 (CPA1969), and anti-Strep tag (CPA9045) antibodies from Cohesion Biosciences (China); anti-FLAG (0912–1) and anti-iNOS (RT1332) antibodies from HuaAn Biotechnology (China); anti-GAPDH (abs830030) antibody from Absin Bioscience (China).

Techniques: Quantitative Proteomics, Sequencing, Modification, Tandem Mass Spectroscopy, Labeling, Over Expression, Transfection, Plasmid Preparation, Western Blot, Knockdown, Mutagenesis, Knock-Out

A proposed model for regulation of the inflammatory response by CRBN. CRBN assembles to an active E3 ligase with DDB1, Cul4A, and ROC1; promotes c-Jun ubiquitination; and mediates its degradation by the 26S proteasome. The reduction of c-Jun protein diminishes AP-1 transcriptional activity and thus reduces LPS-stimulated expression of inflammatory cytokines such as iNOS, COX-2, IL-1β, IL-6, etc.

Journal: The Journal of Biological Chemistry

Article Title: Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun

doi: 10.1074/jbc.RA118.002246

Figure Lengend Snippet: A proposed model for regulation of the inflammatory response by CRBN. CRBN assembles to an active E3 ligase with DDB1, Cul4A, and ROC1; promotes c-Jun ubiquitination; and mediates its degradation by the 26S proteasome. The reduction of c-Jun protein diminishes AP-1 transcriptional activity and thus reduces LPS-stimulated expression of inflammatory cytokines such as iNOS, COX-2, IL-1β, IL-6, etc.

Article Snippet: Antibodies used in this work were from the following companies: anti-HA (sc-7392) and anti-ubiquitin (Ub, sc-8017) antibodies from Santa Cruz Biotechnology (USA); anti-Cul4A (2699S) antibody from Cell Signaling Technology (USA); anti-c-Jun (CPA1634), anti-COX-2 (CPA1969), and anti-Strep tag (CPA9045) antibodies from Cohesion Biosciences (China); anti-FLAG (0912–1) and anti-iNOS (RT1332) antibodies from HuaAn Biotechnology (China); anti-GAPDH (abs830030) antibody from Absin Bioscience (China).

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing

E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, Cul4a, and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: E1, E2, and E3 interacting with the ACR, Ddb1, Crbn, Stub1, Cul4a, and Cul4b, are the most abundant UPS-linked proteins that bind to the ACR Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); and the NSAF (4th to 8th columns. Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of these five proteins.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

Stub1 and CRL4CRBN E3 ligases bind distinct regions of the ACR. A, schematic diagrams of the structural domains of the ACR. Jcasp and Ccas are generated by a double γ-secretase and caspase cleavage of APP. The Thr668 and Tyr682 residues that are phosphorylated are underlined. B, Western blotting analysis of pulldowns shows that Crbn, Ddb1, Cul4a, specifically bind St-Ccas, St-CcasTyr(P), and St-CcasThr(P). Stub1 binds specifically St-JCasp. Two previously known APP interactors, Grb2 and Pin1, bind St-CcasTyr(P) and St-CcasThr(P), respectively. This evidence validates the proteomic approach used. In. indicates the input. The WB shown are representative of at least three independent experiments.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Stub1 and CRL4CRBN E3 ligases bind distinct regions of the ACR. A, schematic diagrams of the structural domains of the ACR. Jcasp and Ccas are generated by a double γ-secretase and caspase cleavage of APP. The Thr668 and Tyr682 residues that are phosphorylated are underlined. B, Western blotting analysis of pulldowns shows that Crbn, Ddb1, Cul4a, specifically bind St-Ccas, St-CcasTyr(P), and St-CcasThr(P). Stub1 binds specifically St-JCasp. Two previously known APP interactors, Grb2 and Pin1, bind St-CcasTyr(P) and St-CcasThr(P), respectively. This evidence validates the proteomic approach used. In. indicates the input. The WB shown are representative of at least three independent experiments.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Generated, Western Blot

Ddb1, Crbn, Cul4a, and Cul4b bind to Ccas and Stub1 binds to JCasp Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of Ddb1, Crbn, Cul4a, and Cul4b, whereas binding of Pin1 and Grb2 is dependent on phosphorylation of Thr 668 and Tyr 682 , respectively. Several of the other UPS-related proteins that are bound to the ACR bind to either Ccas or JCasp or both.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Ddb1, Crbn, Cul4a, and Cul4b bind to Ccas and Stub1 binds to JCasp Phosphorylation of Thr 668 and/or Tyr 682 does not significantly alter binding of Ddb1, Crbn, Cul4a, and Cul4b, whereas binding of Pin1 and Grb2 is dependent on phosphorylation of Thr 668 and Tyr 682 , respectively. Several of the other UPS-related proteins that are bound to the ACR bind to either Ccas or JCasp or both.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

Binding of Ddb1, Cul4a, and Cul4b to ACR Thr(P)Tyr(P) requires Crbn Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown from WT mouse brains (4th column); NSAF of pulldown from Crbn -KO mouse brains (5th column). Binding of Stub1, Grb2, and Pin1 is independent of Crbn.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: Binding of Ddb1, Cul4a, and Cul4b to ACR Thr(P)Tyr(P) requires Crbn Table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown from WT mouse brains (4th column); NSAF of pulldown from Crbn -KO mouse brains (5th column). Binding of Stub1, Grb2, and Pin1 is independent of Crbn.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Binding Assay

AL2CR, but not the AL1CR, binds CRL4CRBN via the substrate recognition pocket of Crbn. A, alignments of the ACR, AL1CR, and AL2CR show that the ACR and the AL2CR are the most conserved intracellular regions of the APP protein family. B, Western blotting analysis of pulldowns with brains isolated from WT mice shows that the AL2CR, but not the AL1CR, interacts with Cul4a, Ddb1, and Crbn. The WB shown is representative of two independent experiments. C, St-AL2CR binds Ddb1 in lysate from WT but not from Crbn-KO mice. The WB shown is representative of three independent experiments. D, Western blotting analysis of St-AL2CR pulldowns from brains isolated from WT mice shows that incubation of the lysates with either thalidomide and lenalidomide prior to pulldowns interferes with the AL2CR-Crbn interaction. The WB shown is representative of two independent experiments.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: AL2CR, but not the AL1CR, binds CRL4CRBN via the substrate recognition pocket of Crbn. A, alignments of the ACR, AL1CR, and AL2CR show that the ACR and the AL2CR are the most conserved intracellular regions of the APP protein family. B, Western blotting analysis of pulldowns with brains isolated from WT mice shows that the AL2CR, but not the AL1CR, interacts with Cul4a, Ddb1, and Crbn. The WB shown is representative of two independent experiments. C, St-AL2CR binds Ddb1 in lysate from WT but not from Crbn-KO mice. The WB shown is representative of three independent experiments. D, Western blotting analysis of St-AL2CR pulldowns from brains isolated from WT mice shows that incubation of the lysates with either thalidomide and lenalidomide prior to pulldowns interferes with the AL2CR-Crbn interaction. The WB shown is representative of two independent experiments.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: Western Blot, Isolation, Incubation

UPS-related proteins, including Ddb1, Crbn, Stub1, Cul4a, and Cul4b, bind to the AL2CR but not to the AL1CR The table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown of WT mouse brains with St alone (4th column); St-AL1CR (5th column); and NSAF of pulldown of WT mouse brains with St-AL2CR (6th column).

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: UPS-related proteins, including Ddb1, Crbn, Stub1, Cul4a, and Cul4b, bind to the AL2CR but not to the AL1CR The table contains the list of proteins identified (1st column); the database accession numbers (2nd column); the molecular mass in kDa (3rd column); NSAF of pulldown of WT mouse brains with St alone (4th column); St-AL1CR (5th column); and NSAF of pulldown of WT mouse brains with St-AL2CR (6th column).

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques:

In vitro ubiquitination of proteins present in the St-ACR Tyr(P)Thr(P) pulldowns 1st column lists some of the proteins ubiquitinated in vitro in an ACR Tyr(P)Thr(P) -dependent manner. 2nd column lists all the lysine residues found ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in previous UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported online. 3rd column reports database accession numbers.

Journal: The Journal of Biological Chemistry

Article Title: Amyloid Precursor Protein (APP) May Act as a Substrate and a Recognition Unit for CRL4 CRBN and Stub1 E3 Ligases Facilitating Ubiquitination of Proteins Involved in Presynaptic Functions and Neurodegeneration *

doi: 10.1074/jbc.M116.733626

Figure Lengend Snippet: In vitro ubiquitination of proteins present in the St-ACR Tyr(P)Thr(P) pulldowns 1st column lists some of the proteins ubiquitinated in vitro in an ACR Tyr(P)Thr(P) -dependent manner. 2nd column lists all the lysine residues found ubiquitinated in vitro. The K-ub found in our UbiScans from mouse brains are indicated with (m*) and in previous UbiScan experiments from mouse (m), human (h), rat (r) and chicken (c) tissues, which are reported online. 3rd column reports database accession numbers.

Article Snippet: The following antibodies were used in WB: anti-Ddb1 (catalog no. 6998s, lot 1, rabbit monoclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Pin-1 (catalog no. 3722s, lot 3, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Stub1 (catalog no. 2080s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:300); anti-Grb2 (catalog no. 3972s, lot 2, rabbit polyclonal antibody, Cell Signaling Technology, dilution 1:1000); anti-Cul4a (catalog no. 14851-1-AP, lot 1, rabbit polyclonal antibody, ProteinTech, dilution 1:300); anti-Crbn (catalog no. HPA045910, lot Q103829, rabbit polyclonal antibody, Sigma, dilution 1:500); and anti-FLAG M2 (catalog no. A2220, lot SLBF8148, mouse monoclonal antibody, Sigma, dilution 1:1000).

Techniques: In Vitro

A GSCs transduced with control or RNAi targeting CDH1, CUL4A, UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.

Journal: Nature Communications

Article Title: Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma

doi: 10.1038/s41467-021-26653-6

Figure Lengend Snippet: A GSCs transduced with control or RNAi targeting CDH1, CUL4A, UBR5, and WWP2 were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. B GSCs transduced with WWP2 RNAi or control were harvested 6 days later and subjected to immunoblotting. Data were representative of three independent experiments. C Cells isolated from patient tumors were subjected to scRNA-seq. Malignant cells were identified by expression-based inference of copy number variation as in Fig. . Cells are colored by expression level for WWP2. D GSC lysates were subjected to anti-SOX2 immunoprecipitation (IP). Input lysates and IP samples were subjected to immunoblotting. Data were representative of three independent experiments. E GSCs transduced with lentiviruses to stably express 6X-Histidine epitope-tagged SOX2 and FLAG epitope-tagged ubiquitin were transduced with WWP2 RNAi or control. Lysates were subjected to IP under denaturing conditions using nickel-NTA beads. IP and input samples were subjected to immunoblotting. Data were representative of three independent experiments. F GSCs transduced with WWP2 RNAi or control were subjected to ELDA as in Fig. . Data represent mean ± SEM ( n = 3, unpaired t -test, ** P < 0.01). See Source Data File for exact P values and statistical parameters.

Article Snippet: Antibodies used for this study include monoclonal rabbit anti-SOX2 (3579) (1:1000, Cell Signaling Technology), polyclonal goat anti-SOX2 (sc-17320) (2 μg antibody per 500 μg lysate protein, Santa Cruz Biotechnology), monoclonal rabbit anti-Sox2 SP76 (1:200, Cell Marque), monoclonal rabbit anti-SOX2 D9B8N (2 μg antibody per 500 μg lysate protein, Cell Signaling Technology), monoclonal mouse anti-alpha-Tubulin (1:4000, T5168) (Sigma), monoclonal mouse anti-HA.11 epitope tag (901502) (1:1000, Biolegend), anti-HA, rabbit monoclonal C29F4, (1:1000, Cell Signaling Technology), anti-Myc, mouse monoclonal, clone 4 A6, (1:1000, Millipore-Sigma), anti-Myc, mouse monoclonal, clone 9E10, (2 μg antibody per 500 μg lysate protein, Millipore-Sigma), polyclonal rabbit anti-GFP (A6455) (1:5000, Thermo Fisher), monoclonal mouse anti-GFP (A11120) (1 μg/mL lysate, Thermo Fisher), monoclonal mouse anti-TRIM26 (sc-393832) (1:200, Santa Cruz Biotechnology), monoclonal mouse anti-beta-Actin (sc-47778) (1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-FLAG M2 (F3165) (1:2000, Sigma), polyclonal rabbit anti-His-Tag (1:1000, 2365) (Cell Signaling Technology), monoclonal mouse anti-FZR1(CDH1) (ab3242) (1:500, Abcam), polyclonal rabbit anti-Cullin-4A (1:1000, C0371) (Sigma), polyclonal rabbit anti-EDD1(UBR5) (A300-573A) (1:2000, Bethyl Laboratories), polyclonal rabbit anti-WWP2 (A302-935A) (1:2000, Bethyl Laboratories), monoclonal mouse anti-Human nuclear antigen 235-1 (1:500, Novus Biological), and Normal goat IgG (NI02) (Millipore-Sigma).

Techniques: Transduction, Western Blot, Isolation, Expressing, Immunoprecipitation, Stable Transfection, FLAG-tag