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  • 95
    Thermo Fisher anti xpress antibody
    Anti Xpress Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 768 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti xpress antibody/product/Thermo Fisher
    Average 95 stars, based on 768 article reviews
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    99
    Millipore anti flag m2 antibody
    Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 6290 article reviews
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    anti flag m2 antibody - by Bioz Stars, 2020-12
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    99
    Bio-Rad laemmli sample buffer
    Laemmli Sample Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 4406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/laemmli sample buffer/product/Bio-Rad
    Average 99 stars, based on 4406 article reviews
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    94
    Cell Signaling Technology Inc malt1
    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on <t>Malt1</t> and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file
    Malt1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/malt1/product/Cell Signaling Technology Inc
    Average 94 stars, based on 65 article reviews
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    99
    Cell Signaling Technology Inc stat3 124h6
    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on <t>Malt1</t> and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file
    Stat3 124h6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/stat3 124h6/product/Cell Signaling Technology Inc
    Average 99 stars, based on 20 article reviews
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    99
    GE Healthcare protein a sepharose pas cl 4b
    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on <t>Malt1</t> and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file
    Protein A Sepharose Pas Cl 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/protein a sepharose pas cl 4b/product/GE Healthcare
    Average 99 stars, based on 6 article reviews
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    99
    Thermo Fisher lipofectamine 2000
    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on <t>Malt1</t> and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file
    Lipofectamine 2000, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 466214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 466214 article reviews
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    Image Search Results


    Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on Malt1 and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Hectd3 interacts and promotes non-degradative K27- and K29-linked polyubiquitination on Malt1 and NF-κB activation in CD4 + T cells. a Immunoblot of Hectd3, Malt1, and GAPDH following Malt1 or IgG immunoprecipitation of protein extracts from draining lymph node CD4 + T cells of WT mice 13 days following EAE induction. b Immunoblot of Malt1 following polyubiquitinated protein enrichment of extract from draining lymph node CD4 + T cells of wild-type Hectd3 −/− or (WT) mice 13 days following EAE induction. About 1.5× the amount of total protein from Hectd3 −/− CD4 + T cells, compared to WT CD4 + T cells, was used to normalize for the reduction in Malt1 protein level in Hectd3 −/− CD4 + T cells. At least 700 μg of total protein from mouse primary CD4 + T cells were enriched for ubiquitinated protein with Ubiquitinated Protein Enrichment Kit or Anti-Ub TUBE2, Agarose following manufacturer’s protocol. c , d Percentage of p65 or RelB nuclear translocation using p65/DAPI similarity analysis from ImageStream, in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− and WT mice, 13 days following EAE induction. e , f ImageStream fluorescence imaging of PMA/ionomycin-induced p65 nuclear translocation ( e ) or RelB ( f ) in CD4 + T cells isolated from draining lymph nodes of Hectd3 −/− or WT mice, 13 days following EAE induction. g Immunoblot of HA, Flag, and Xpress following two-step Flag immunoprecipitation of protein extracts from HEK293T cells co-transfected with the indicated HA-Ub K only mutants, Flag-Malt1A, and Xpress-Hectd3. HA-Ub K only mutant denote that the only lysine in the HA-tagged ubiquitin is at the indicated residue, and all other lysine residues are mutated to arginine. HA-Ub K0 mutant indicate that all seven lysine residues are mutated to arginine. a – g file

    Article Snippet: A total of 2 μg of Anti-Flag M2 antibody (Cat#F1804, Sigma, Millipore-Sigma, MO, USA) or Anti-Xpress antibody (Cat#R910-25, Invitrogen, Thermo Fisher Scientific, PA, USA) or Stat3 (124H6) (Cat#9139, Cell Signaling Technology, MA, USA) or Malt1 (Cat#2494, Cell Signaling Technology, MA, USA) was added to pre-cleared samples, and then incubated at 4 °C with rotation for 30 min. A total of 40 μl of 50% PAS was then added to antibody-pre-cleared sample mix and incubated at 4 °C with rotation from 4 h to overnight.

    Techniques: Activation Assay, Immunoprecipitation, Mouse Assay, Protein Enrichment, Translocation Assay, Isolation, Fluorescence, Imaging, Transfection, Mutagenesis

    Ubiquitination of Malt1A K648 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Malt1A, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated DANKGTPEETGSYLVSK peptide (ubiquitinated K648 residue) of Malt1A. Parent ion corresponding to DANkGTPEETGSYLVSK peptide mass has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated. b Representative immunoblot of Flag and ubiquitin following polyubiquitinated protein enrichment of extracts using TUBE2 from CD90.1+ sorted EL4 cells transduced with MSCV-CD90.1-Flag-Malt1A or MSCV-CD90.1-Flag-Malt1A K648R retrovirus. c Flow cytometry analysis of intracellular IL-17A and intranuclear RORγt in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retrovirus and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on CD90.1 + T cells. d MFI of Malt1A in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retroviruses and in vitro polarized under Th17 condition. Data ( n = 6) are mean of three independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t file

    Journal: Nature Communications

    Article Title: Hectd3 promotes pathogenic Th17 lineage through Stat3 activation and Malt1 signaling in neuroinflammation

    doi: 10.1038/s41467-019-08605-3

    Figure Lengend Snippet: Ubiquitination of Malt1A K648 by Hectd3 is essential for RORγt + IL-17A hi Th17 cell generation. a HEK293T cells were co-transfected with HA-Ub, Flag-Malt1A, and Xpress-Hectd3. Extracts were immunoprecipitated with anti-Flag antibodies, followed by trypsin digestion and tandem mass spectrometry, as described in Material and methods. A fragmentation spectrum of ubiquitinated DANKGTPEETGSYLVSK peptide (ubiquitinated K648 residue) of Malt1A. Parent ion corresponding to DANkGTPEETGSYLVSK peptide mass has been subjected to higher-energy collisional dissociation in mass spectrometer. The detected b- and y-fragment ion series have been annotated. b Representative immunoblot of Flag and ubiquitin following polyubiquitinated protein enrichment of extracts using TUBE2 from CD90.1+ sorted EL4 cells transduced with MSCV-CD90.1-Flag-Malt1A or MSCV-CD90.1-Flag-Malt1A K648R retrovirus. c Flow cytometry analysis of intracellular IL-17A and intranuclear RORγt in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retrovirus and in vitro polarized under Th17 conditions. Representative of three independent experiments. Gating strategy was first on CD90.1 + T cells. d MFI of Malt1A in CD90.1 + Malt1 −/− CD4 + T cells transduced with indicated retroviruses and in vitro polarized under Th17 condition. Data ( n = 6) are mean of three independent experiments and are presented as mean ± SEM; p value was obtained from Student’s t file

    Article Snippet: A total of 2 μg of Anti-Flag M2 antibody (Cat#F1804, Sigma, Millipore-Sigma, MO, USA) or Anti-Xpress antibody (Cat#R910-25, Invitrogen, Thermo Fisher Scientific, PA, USA) or Stat3 (124H6) (Cat#9139, Cell Signaling Technology, MA, USA) or Malt1 (Cat#2494, Cell Signaling Technology, MA, USA) was added to pre-cleared samples, and then incubated at 4 °C with rotation for 30 min. A total of 40 μl of 50% PAS was then added to antibody-pre-cleared sample mix and incubated at 4 °C with rotation from 4 h to overnight.

    Techniques: Transfection, Immunoprecipitation, Mass Spectrometry, Protein Enrichment, Transduction, Flow Cytometry, Cytometry, In Vitro