zebrafish ubi Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs nebuilder hifi assembly kit
    Nebuilder Hifi Assembly Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nebuilder hifi assembly kit/product/New England Biolabs
    Average 99 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    nebuilder hifi assembly kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Millipore tamoxifen
    Tamoxifen, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17729 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tamoxifen/product/Millipore
    Average 99 stars, based on 17729 article reviews
    Price from $9.99 to $1999.99
    tamoxifen - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    93
    Addgene inc pentr5 ubi
    Pentr5 Ubi, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pentr5 ubi/product/Addgene inc
    Average 93 stars, based on 60 article reviews
    Price from $9.99 to $1999.99
    pentr5 ubi - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    88
    Thermo Fisher tol2kit
    Tol2kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tol2kit/product/Thermo Fisher
    Average 88 stars, based on 159 article reviews
    Price from $9.99 to $1999.99
    tol2kit - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    89
    Thermo Fisher pentr d
    Pentr D, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 549 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pentr d/product/Thermo Fisher
    Average 89 stars, based on 549 article reviews
    Price from $9.99 to $1999.99
    pentr d - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    91
    Cell Signaling Technology Inc h2b
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    H2b, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h2b/product/Cell Signaling Technology Inc
    Average 91 stars, based on 198 article reviews
    Price from $9.99 to $1999.99
    h2b - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc phospho myosin light chain 2 ser19 antibody
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    Phospho Myosin Light Chain 2 Ser19 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho myosin light chain 2 ser19 antibody/product/Cell Signaling Technology Inc
    Average 95 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    phospho myosin light chain 2 ser19 antibody - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    90
    Roche expand high fidelity pcr kit
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    Expand High Fidelity Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand high fidelity pcr kit/product/Roche
    Average 90 stars, based on 736 article reviews
    Price from $9.99 to $1999.99
    expand high fidelity pcr kit - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    88
    Thermo Fisher pentr5
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    Pentr5, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pentr5/product/Thermo Fisher
    Average 88 stars, based on 107 article reviews
    Price from $9.99 to $1999.99
    pentr5 - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    94
    Roche pcr amplification
    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone <t>H2B.</t> A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.
    Pcr Amplification, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 13428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr amplification/product/Roche
    Average 94 stars, based on 13428 article reviews
    Price from $9.99 to $1999.99
    pcr amplification - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    99
    Proteintech tdp 43
    Characterization of <t>TDP-43</t> NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Tdp 43, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 1754 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tdp 43/product/Proteintech
    Average 99 stars, based on 1754 article reviews
    Price from $9.99 to $1999.99
    tdp 43 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Proteintech mouse gapdh monoclonal
    Characterization of <t>TDP-43</t> NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Mouse Gapdh Monoclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 1612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse gapdh monoclonal/product/Proteintech
    Average 99 stars, based on 1612 article reviews
    Price from $9.99 to $1999.99
    mouse gapdh monoclonal - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho erk1 2
    Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by <t>Erk1/2</t> depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h
    Phospho Erk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 9805 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho erk1 2/product/Cell Signaling Technology Inc
    Average 99 stars, based on 9805 article reviews
    Price from $9.99 to $1999.99
    phospho erk1 2 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Proteintech rabbit gapdh polyclonal
    Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by <t>Erk1/2</t> depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h
    Rabbit Gapdh Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 1654 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit gapdh polyclonal/product/Proteintech
    Average 99 stars, based on 1654 article reviews
    Price from $9.99 to $1999.99
    rabbit gapdh polyclonal - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    96
    Proteintech rabbit tdp 43 c terminal polyclonal
    Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by <t>Erk1/2</t> depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h
    Rabbit Tdp 43 C Terminal Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 96/100, based on 207 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit tdp 43 c terminal polyclonal/product/Proteintech
    Average 96 stars, based on 207 article reviews
    Price from $9.99 to $1999.99
    rabbit tdp 43 c terminal polyclonal - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    93
    Cell Signaling Technology Inc histone h2a
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Histone H2a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/histone h2a/product/Cell Signaling Technology Inc
    Average 93 stars, based on 244 article reviews
    Price from $9.99 to $1999.99
    histone h2a - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    94
    TaKaRa subcloning
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Subcloning, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 902 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/subcloning/product/TaKaRa
    Average 94 stars, based on 902 article reviews
    Price from $9.99 to $1999.99
    subcloning - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    90
    ZIRC Inc 1st mouse anti zns 5
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    1st Mouse Anti Zns 5, supplied by ZIRC Inc, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1st mouse anti zns 5/product/ZIRC Inc
    Average 90 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    1st mouse anti zns 5 - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    94
    TaKaRa pegfp n3
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Pegfp N3, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 3038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pegfp n3/product/TaKaRa
    Average 94 stars, based on 3038 article reviews
    Price from $9.99 to $1999.99
    pegfp n3 - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    egfp  (TaKaRa)
    94
    TaKaRa egfp
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 8760 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/egfp/product/TaKaRa
    Average 94 stars, based on 8760 article reviews
    Price from $9.99 to $1999.99
    egfp - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    99
    TaKaRa 1st rabbit anti mcherry dsred
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    1st Rabbit Anti Mcherry Dsred, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1st rabbit anti mcherry dsred/product/TaKaRa
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    1st rabbit anti mcherry dsred - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    92
    Addgene inc mitfa promoter
    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone <t>H2A</t> as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.
    Mitfa Promoter, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mitfa promoter/product/Addgene inc
    Average 92 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    mitfa promoter - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone H2B. A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquitin-specific Peptidase 42 (USP42) Functions to Deubiquitylate Histones and Regulate Transcriptional Activity *

    doi: 10.1074/jbc.M114.589267

    Figure Lengend Snippet: The linker and lysine-rich domain of USP42 are necessary for appropriate localization of USP42 and interaction of histone H2B. A , endogenous coimmunoprecipitation showing specific interaction of USP42 and H2B. B , schematic representation of the USP42 overexpression mutants used in subsequent experiments. C , immunoprecipitation of GFP-USP42 mutants to determine their interaction with H2B. Although the catalytic activity of USP42 is not required to bind H2B, deletion of the linker domain causes loss of H2B association, and deletion of the lysine-rich domain leads to a reduction of interaction. D , confocal immunofluorescence visualizing the localization of GFP-USP42 wild type and mutant proteins. Although the catalytic activity of USP42 does not alter the localization of USP42, deletion of the linker domain causes accumulation in the nucleoli, and deletion of the lysine-rich domain leads to a diffuse nuclear localization. E , in vitro deubiquitylation assay of Ubi-H2B by USP42. Wild type USP42 efficiently deubiquitylates Ubi-H2B. Disruption of the DUB domain renders USP42 inactive toward Ubi-H2B. ΔKK mutant and Δlinker mutants, which both harbor an intact DUB domain, retain the ability to deubiquitylate histone H2B. F , quantification of E . Ubi-H2B Western blots shown in E were quantified using the LI-COR Biosciences Odyssey. G, In vitro deubiquitylation assay of Ubi-H2B and Ubi-H2A by USP42. H , depletion of USP42 leads to an increase in H2B ubiquitylation and does not alter H2A ubiquitination. LI-COR quantification showing levels of H2B and H2A ubiquitylation ( Ubi-Histone ) normalized to unmodified histone with and without USP42 knockdown. Error bars represent the S.D. of three independent replicas. IP , immunoprecipitation; CTL , control.

    Article Snippet: Antibodies used were: USP42 (Atlas, HPA006752), GFP (Abcam, ab6556; Roche Applied Science, 11814460001), H2B (Cell Signaling Technology, 2934), RNA Pol II (Santa Cruz, SC-65884), actin (Santa Cruz, SC-1616), tubulin (Santa Cruz, SC-8035), Ubi-H2B (Cell Signaling Technology, 5546), H2A (Cell Signaling Technology, 3636), and Ubi-H2A (Millipore, 05-678; Cell Signaling Technology, 8240).

    Techniques: Over Expression, Immunoprecipitation, Activity Assay, Immunofluorescence, Mutagenesis, In Vitro, Western Blot, CTL Assay

    USP42 knockdown increases endogenous H2B ubiquitylation specifically on the start and early extension sites of the p21 promoter upon its induction. A–D , chromatin immunoprecipitations showing the ubiquitylation status of H2B and H2A on the p21 gene following USP42 knockdown. H2B ubiquitylation ( ubi-H2B ) increases upon p21 induction at the initiator and the first intron. This is further increased by knockdown of USP42 ( A ), whereas H2A ubiquitylation ( ubi-H2A ) is not influenced by p21 induction or USP42 knockdown ( B ). The difference in ubiquitylation is not an indirect result of general histone deposition because H2B and H2A levels are not altered ( C and D ). Error bars represent the S.D. of three independent replicas. E , expression analysis of p21 mRNA by quantitative RT-PCR. Knockdown of USP42 decreases p21 mRNA up to 38 h. F , chromatin immunoprecipitation showing the recruitment of RNA Pol II to the p21 gene upon p53 induction. USP42 knockdown ( red ) increases RNA Pol II levels closer to the start site of transcription, whereas a reduced amount of RNA Pol II is observed in the distal part of the gene relative to control ( blue ). Error bars represent the S.D. of three independent replicas. ActD , actinomycin D; CTL , control; rel. , relative; p53BS , p53 binding site.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquitin-specific Peptidase 42 (USP42) Functions to Deubiquitylate Histones and Regulate Transcriptional Activity *

    doi: 10.1074/jbc.M114.589267

    Figure Lengend Snippet: USP42 knockdown increases endogenous H2B ubiquitylation specifically on the start and early extension sites of the p21 promoter upon its induction. A–D , chromatin immunoprecipitations showing the ubiquitylation status of H2B and H2A on the p21 gene following USP42 knockdown. H2B ubiquitylation ( ubi-H2B ) increases upon p21 induction at the initiator and the first intron. This is further increased by knockdown of USP42 ( A ), whereas H2A ubiquitylation ( ubi-H2A ) is not influenced by p21 induction or USP42 knockdown ( B ). The difference in ubiquitylation is not an indirect result of general histone deposition because H2B and H2A levels are not altered ( C and D ). Error bars represent the S.D. of three independent replicas. E , expression analysis of p21 mRNA by quantitative RT-PCR. Knockdown of USP42 decreases p21 mRNA up to 38 h. F , chromatin immunoprecipitation showing the recruitment of RNA Pol II to the p21 gene upon p53 induction. USP42 knockdown ( red ) increases RNA Pol II levels closer to the start site of transcription, whereas a reduced amount of RNA Pol II is observed in the distal part of the gene relative to control ( blue ). Error bars represent the S.D. of three independent replicas. ActD , actinomycin D; CTL , control; rel. , relative; p53BS , p53 binding site.

    Article Snippet: Antibodies used were: USP42 (Atlas, HPA006752), GFP (Abcam, ab6556; Roche Applied Science, 11814460001), H2B (Cell Signaling Technology, 2934), RNA Pol II (Santa Cruz, SC-65884), actin (Santa Cruz, SC-1616), tubulin (Santa Cruz, SC-8035), Ubi-H2B (Cell Signaling Technology, 5546), H2A (Cell Signaling Technology, 3636), and Ubi-H2A (Millipore, 05-678; Cell Signaling Technology, 8240).

    Techniques: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, CTL Assay, Binding Assay

    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

    doi: 10.1074/jbc.RA118.003440

    Figure Lengend Snippet: Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.

    Article Snippet: The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1).

    Techniques: Mutagenesis, Mass Spectrometry, Plasmid Preparation, Inhibition, Lysis, Affinity Purification, Western Blot, Staining, Immunolabeling, Fractionation, Marker

    Cross-talk between C-terminal ubiquitinylation and phosphorylation of CTF. HEK293E cells were double-transfected with CTF WT , CTF 4×KR , CTF K408R , and the phospho-mimic- or -dead CTF SSDD or CTF SSAA double mutants (all CTF constructs fused to mCherry) or mCherry-control vector (−). After cell lysis with urea buffer, His 6 -ubiquitinylated proteins were affinity-purified with Ni-NTA–agarose. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis with antibodies detecting mCherry, TDP-43, ubiquitin, His 6 , phospho-TDP-43, and GAPDH as loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

    doi: 10.1074/jbc.RA118.003440

    Figure Lengend Snippet: Cross-talk between C-terminal ubiquitinylation and phosphorylation of CTF. HEK293E cells were double-transfected with CTF WT , CTF 4×KR , CTF K408R , and the phospho-mimic- or -dead CTF SSDD or CTF SSAA double mutants (all CTF constructs fused to mCherry) or mCherry-control vector (−). After cell lysis with urea buffer, His 6 -ubiquitinylated proteins were affinity-purified with Ni-NTA–agarose. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis with antibodies detecting mCherry, TDP-43, ubiquitin, His 6 , phospho-TDP-43, and GAPDH as loading control.

    Article Snippet: The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1).

    Techniques: Transfection, Construct, Plasmid Preparation, Lysis, Affinity Purification, Western Blot

    Investigation of ubiquitinylation sites for the hyper-ubiquitinylated mutant TDP-43 K263E . HEK293E cells were transfected with FLAG–TDP-43 WT and FLAG–TDP-43 K263E alone or combined with the indicated lysine substitutions localized in the C-terminal part ( A ) or the NLS sequence ( B ). His 6 -ubiquitin (+) or vector control (−) was cotransfected, as indicated. Cells were then treated with MG-132 (+) or DMSO (−) for 2 h. The urea-soluble lysates were prepared, and His 6 -ubiquitin–conjugated proteins were pulled down from cell lysates. Total cell lysates ( Input ) and Ni-NTA–agarose eluates were analyzed with Western blotting and stained for FLAG, TDP-43, ubiquitin, and GAPDH, and in B additionally with anti-His 6 . Lysates samples in B were re-run and subjected to Western blot analysis of phospho-TDP-43. C, HEK293E cells overexpressing FLAG–TDP-43 WT and FLAG–TDP-43 K263E alone or combined with the indicated Lys-84 and Lys-94 substitutions were immunostained with rabbit anti-FLAG ( green ). Nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars correspond to 20 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

    doi: 10.1074/jbc.RA118.003440

    Figure Lengend Snippet: Investigation of ubiquitinylation sites for the hyper-ubiquitinylated mutant TDP-43 K263E . HEK293E cells were transfected with FLAG–TDP-43 WT and FLAG–TDP-43 K263E alone or combined with the indicated lysine substitutions localized in the C-terminal part ( A ) or the NLS sequence ( B ). His 6 -ubiquitin (+) or vector control (−) was cotransfected, as indicated. Cells were then treated with MG-132 (+) or DMSO (−) for 2 h. The urea-soluble lysates were prepared, and His 6 -ubiquitin–conjugated proteins were pulled down from cell lysates. Total cell lysates ( Input ) and Ni-NTA–agarose eluates were analyzed with Western blotting and stained for FLAG, TDP-43, ubiquitin, and GAPDH, and in B additionally with anti-His 6 . Lysates samples in B were re-run and subjected to Western blot analysis of phospho-TDP-43. C, HEK293E cells overexpressing FLAG–TDP-43 WT and FLAG–TDP-43 K263E alone or combined with the indicated Lys-84 and Lys-94 substitutions were immunostained with rabbit anti-FLAG ( green ). Nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars correspond to 20 μm.

    Article Snippet: The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1).

    Techniques: Mutagenesis, Transfection, Sequencing, Plasmid Preparation, Western Blot, Staining

    Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by Erk1/2 depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h

    Journal: Cell Death & Disease

    Article Title: XAF1 forms a positive feedback loop with IRF-1 to drive apoptotic stress response and suppress tumorigenesis

    doi: 10.1038/s41419-018-0867-4

    Figure Lengend Snippet: Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by Erk1/2 depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h

    Article Snippet: Antibodies specific for XAF1 (SC-19194, ab17204), IRF-1 (SC-497, SC-514934), IRF-3 (SC-9082), IRF-7 (SC-9083), CHIP (SC-33264, SC-66830), cleaved PARP (CST #9541), cleaved CASP3 (CST #9661S), p65/RelA (SC-372, SC-514451), MMP-9 (CST #3852), phospho-Erk1/2 (CST #9101), U1 SnRNP 70 (SC-9571), anti-HA (SC-7392, SC-805, A2095), anti-GFP (SC-9996, ab69314), anti-Flag (SC-166384), anti-V5 (SC-83849, A7345), anti-Myc (SC-40), anti-His (SC-803), anti-GST(SC-33613), anti-Ubi (CST #8081), and β-tubulin (T0198) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling Technology, Abcam (Cambridge, MA, USA), or BD Bioscience (Franklin Lakes, NJ, USA).

    Techniques: Expressing, Immunohistochemistry, Staining, Transfection, Incubation

    Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by Erk1/2 depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h

    Journal: Cell Death & Disease

    Article Title: XAF1 forms a positive feedback loop with IRF-1 to drive apoptotic stress response and suppress tumorigenesis

    doi: 10.1038/s41419-018-0867-4

    Figure Lengend Snippet: Frequent alteration of the IRF-1−XAF1 loop in human cancer. a , b A tight correlation of XAF1 and IRF-1 expression in human cancer cells. Relative expression levels were classified as levels 0−5. r Pearson’s correlation coefficient. c , d IHC study showing a strong correlation of XAF1 and IRF-1 immunoreactivity (dark brown) in human colon carcinoma and normal tissues. Purple staining indicates the nuclei. Relative staining levels were classified as levels 0–5. r Pearson’s correlation coefficient. e Ras suppression of IFN-mediated IRF-1 induction in a XAF1-dependent manner. Cells transfected with oncogenic H-Ras (G12V) were exposed to IFN-β (200 U/ml) for 48 h. f IRF-1 elevation by UO126 (10 μM, 8 h) and its blockade by XAF1 depletion. g IRF-1 induction by Erk1/2 depletion in an XAF1-dependent manner. IB assay was performed after 48 h si-Erk1/2 transfection. h Suppression of etoposide-mediated XAF1 and IRF-1 induction by Ras/MEK-activating growth factors. DU145 cells were incubated with EGF (50 ng/ml), IGF (100 ng/ml), FGF (50 ng/ml), or TGF-β1 (2 ng/ml) for 30 min and then exposed to etoposide (50 μM) for 16 h

    Article Snippet: Antibodies specific for XAF1 (SC-19194, ab17204), IRF-1 (SC-497, SC-514934), IRF-3 (SC-9082), IRF-7 (SC-9083), CHIP (SC-33264, SC-66830), cleaved PARP (CST #9541), cleaved CASP3 (CST #9661S), p65/RelA (SC-372, SC-514451), MMP-9 (CST #3852), phospho-Erk1/2 (CST #9101), U1 SnRNP 70 (SC-9571), anti-HA (SC-7392, SC-805, A2095), anti-GFP (SC-9996, ab69314), anti-Flag (SC-166384), anti-V5 (SC-83849, A7345), anti-Myc (SC-40), anti-His (SC-803), anti-GST(SC-33613), anti-Ubi (CST #8081), and β-tubulin (T0198) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling Technology, Abcam (Cambridge, MA, USA), or BD Bioscience (Franklin Lakes, NJ, USA).

    Techniques: Expressing, Immunohistochemistry, Staining, Transfection, Incubation

    Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

    doi: 10.1074/jbc.RA118.003440

    Figure Lengend Snippet: Characterization of TDP-43 NLS lysine mutant ubiquitinylation and subcellular localization. A, schematic overview of TDP-43 ubiquitinylated lysine ( K ) residues that were identified by MS. All other lysine residues are indicated with dots. B, His 6 -ubiquitin (+) or vector control (−) and FLAG–TDP-43 WT , NLS lysine mutants, or FLAG control vector ( Ø ) were overexpressed in HEK293E cells. Proteasomal inhibition with MG-132 for 2 h was followed by cell lysis in urea buffer and affinity purification of His 6 -ubiquitinylated proteins. Total protein ( Input ) and Ni-NTA–agarose eluates ( His 6 Pulldown ) were subjected to Western blot analysis and stained with antibodies against FLAG, TDP-43, His 6 , and GAPDH. C, HEK293E cells overexpressing FLAG–TDP-43 WT and NLS lysine mutants were immunolabeled with mouse anti-FLAG ( green ) and rabbit anti-TDP-43 ( red ), and cell nuclei were counterstained with Hoechst 33342 ( blue ). Scale bars are 20 μm. D, nuclear–cytoplasmic fractionation was performed on HEK293E cells that overexpressed FLAG–TDP-43 WT or the indicated NLS lysine mutants. Cytoplasmic ( cyto ), nuclear ( nu ), and total lysates were analyzed by Western blotting with antibodies detecting FLAG, TDP-43, and Hsp90 as cytoplasmic marker, and histone H2A as nuclear marker. Asterisks label endogenous TDP-43. E, quantification of TDP-43 ( upper graph ) and FLAG ( lower graph ) signals from at least n = 3 experiments as in D . Band intensities were normalized to the FLAG–TDP-43 WT signal of the respective fraction. Data represent the mean ± S.D. *, p ≤ 0.05; ***, p ≤ 0.005.

    Article Snippet: The following antibodies were used in this study for Western blotting (WB) or immunofluorescence stainings (IF): mouse anti-β-actin (WB 1:50.000; catalog no. A5441, Sigma, clone AC-15); mouse anti-GAPDH (WB 1:35.000; catalog no. H86504M, Biodesign International, clone 6C5); mouse anti-FLAG (IF 1:1000; Sigma, clone M2 affinity-purified); rabbit anti-FLAG (IF 1:500; catalog no. 2368, Cell Signaling); peroxidase-conjugated anti-FLAG (WB 1:2000–1:60,000; catalog no. A8592, Sigma, clone M2); mouse anti-His6 (WB 1:3000; catalog no. 27-4710-01, Amersham Biosciences); mouse anti-His6 (WB 1:5.000–10.000; catalog no. MA1-21315, Invitrogen, clone HIS.H8); rabbit polyclonal against histone H2A (WB 1:1000; catalog no. 2578, Cell Signaling); rabbit anti-Hsp90 (WB 1:10,000; catalog no. SPA-846, Stressgen); rabbit anti-eIF2α (WB 1:2000; catalog no. 9722, Cell Signaling); rabbit anti-phospho-eIF2α (WB 1:2000; catalog no. ab4837, Abcam, clone S51); rabbit anti-PARP (WB 1:4000; catalog no. 9542, Cell Signaling); rabbit anti-dsRed “mCherry” (WB 1:5000; catalog no. 632496, Clontech); rabbit anti-TDP-43 (WB 1:10,000, IF 1:1000; catalog no. 10782-2-AP, ProteinTech Group); rat anti-phospho-TDP-43 (Ser-409/410) (WB 1:10, IF 1:50; M. Neumann, clone 1D3); mouse anti-ubiquitin (mono- and poly-) (WB 1:4000; catalog no. MAB1510, Millipore, clone Ubi-1).

    Techniques: Mutagenesis, Mass Spectrometry, Plasmid Preparation, Inhibition, Lysis, Affinity Purification, Western Blot, Staining, Immunolabeling, Fractionation, Marker