zebrafish ubi Search Results


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  • 99
    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    98
    Proteintech anti tdp 43 antibodies
    Znf179-mediates <t>TDP-43</t> polyubiquitination in vitro and in vivo . a and b The total brain lysates of wild-type or Znf179-knockout mice ( a) or 293 T cells transfected with Flag-mZnf179 and treated with 10 μM MG132 for 4 h ( b ) were immunoprecipitated by anti-TDP-43 antibody and analyzed by Western blotting with anti-ubiquitin or anti-TDP-43 antibodies. c , d and e For in vitro polyubiquitination assays, endogenous TDP-43 that was immunoprecipitated with anti-TDP-43 antibody from non-treated N2a cells lysates was included in a mixture of purified E1, ubiquitin, Mg 2+ -ATP, UbcH5c E2 conjugating enzyme, and the E3 ligase, Znf179. The Znf179 E3 ligase was immunoprecipitated from N2a cells stably expressing GFP-mZnf179 ( c ) or from 293 T cells transiently transfected with Flag-mZnf179 or with Flag-mZnf179-5A mutant ( d and e ). The ubiquitination levels of TDP-43 were analyzed by anti-TDP-43 antibody. Data were presented as the mean ± SEM ( n = 3) (*** p
    Anti Tdp 43 Antibodies, supplied by Proteintech, used in various techniques. Bioz Stars score: 98/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Novus Biologicals anti lc3
    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to <t>LC3‐II</t> were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    Anti Lc3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 96/100, based on 425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc entry 2a mcherry
    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to <t>LC3‐II</t> were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    Entry 2a Mcherry, 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
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    99
    Novus Biologicals lc3b antibody
    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to <t>LC3‐II</t> were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    Lc3b Antibody, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 99/100, based on 1583 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc mitfa promoter
    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to <t>LC3‐II</t> were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    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
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    88
    Thermo Fisher pdest tol2 pa2
    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to <t>LC3‐II</t> were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).
    Pdest Tol2 Pa2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Addgene inc pentr5 ubi
    Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), <t>unc503</t> ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).
    Pentr5 Ubi, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher tol2kit
    Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), <t>unc503</t> ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).
    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
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    99
    TaKaRa 1st rabbit anti mcherry dsred
    Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), <t>unc503</t> ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).
    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
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    90
    ZIRC Inc 1st mouse anti zns 5
    Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), <t>unc503</t> ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).
    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
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    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
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    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
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    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
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    99
    Proteintech rabbit gapdh polyclonal
    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.
    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
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    Proteintech rabbit tdp 43 polyclonal
    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.
    Rabbit Tdp 43 Polyclonal, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 791 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    99
    Cell Signaling Technology Inc d9e
    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.
    D9e, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher f ab 2 goat anti rabbit igg h l cross adsorbed secondary antibody
    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.
    F Ab 2 Goat Anti Rabbit Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lr clonase ii plus
    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.
    Lr Clonase Ii Plus, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lr clonase ii plus/product/Thermo Fisher
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    Image Search Results


    Znf179-mediates TDP-43 polyubiquitination in vitro and in vivo . a and b The total brain lysates of wild-type or Znf179-knockout mice ( a) or 293 T cells transfected with Flag-mZnf179 and treated with 10 μM MG132 for 4 h ( b ) were immunoprecipitated by anti-TDP-43 antibody and analyzed by Western blotting with anti-ubiquitin or anti-TDP-43 antibodies. c , d and e For in vitro polyubiquitination assays, endogenous TDP-43 that was immunoprecipitated with anti-TDP-43 antibody from non-treated N2a cells lysates was included in a mixture of purified E1, ubiquitin, Mg 2+ -ATP, UbcH5c E2 conjugating enzyme, and the E3 ligase, Znf179. The Znf179 E3 ligase was immunoprecipitated from N2a cells stably expressing GFP-mZnf179 ( c ) or from 293 T cells transiently transfected with Flag-mZnf179 or with Flag-mZnf179-5A mutant ( d and e ). The ubiquitination levels of TDP-43 were analyzed by anti-TDP-43 antibody. Data were presented as the mean ± SEM ( n = 3) (*** p

    Journal: Journal of Biomedical Science

    Article Title: Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

    doi: 10.1186/s12929-018-0479-4

    Figure Lengend Snippet: Znf179-mediates TDP-43 polyubiquitination in vitro and in vivo . a and b The total brain lysates of wild-type or Znf179-knockout mice ( a) or 293 T cells transfected with Flag-mZnf179 and treated with 10 μM MG132 for 4 h ( b ) were immunoprecipitated by anti-TDP-43 antibody and analyzed by Western blotting with anti-ubiquitin or anti-TDP-43 antibodies. c , d and e For in vitro polyubiquitination assays, endogenous TDP-43 that was immunoprecipitated with anti-TDP-43 antibody from non-treated N2a cells lysates was included in a mixture of purified E1, ubiquitin, Mg 2+ -ATP, UbcH5c E2 conjugating enzyme, and the E3 ligase, Znf179. The Znf179 E3 ligase was immunoprecipitated from N2a cells stably expressing GFP-mZnf179 ( c ) or from 293 T cells transiently transfected with Flag-mZnf179 or with Flag-mZnf179-5A mutant ( d and e ). The ubiquitination levels of TDP-43 were analyzed by anti-TDP-43 antibody. Data were presented as the mean ± SEM ( n = 3) (*** p

    Article Snippet: Five-hundred micrograms of total protein were mixed with 2 μl anti-Znf179 or anti-TDP-43 antibodies (Proteintech Group Cat# 12892–1-AP RRID:AB_2200505) and added to the Antibody Capture Affinity Ligand in 1× Wash Buffer to make a final volume of 500 ul.

    Techniques: In Vitro, In Vivo, Knock-Out, Mouse Assay, Transfection, Immunoprecipitation, Western Blot, Purification, Stable Transfection, Expressing, Mutagenesis

    Znf179 enhances the degradation rate of TDP-43 protein and alters the solubility of TDP-43. a and b Endogenous TDP-43 ( a ) or Myc-hTDP-43 ( b ) in N2a cells with or without stably expressed GFP-mZnf179 was treated with cycloheximide (20 mg/ml) for different time periods. The protein levels of the Myc-hTDP-43 were analyzed by western blotting with anti-TDP-43 or anti-Myc antibody. The graph below the blots showed a quantification of the relative values at each time point, from which the half-lives of the proteins were estimated. Half-life was calculated by linear regression. Data were presented as the mean ± SEM ( n = 3) (*** p

    Journal: Journal of Biomedical Science

    Article Title: Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

    doi: 10.1186/s12929-018-0479-4

    Figure Lengend Snippet: Znf179 enhances the degradation rate of TDP-43 protein and alters the solubility of TDP-43. a and b Endogenous TDP-43 ( a ) or Myc-hTDP-43 ( b ) in N2a cells with or without stably expressed GFP-mZnf179 was treated with cycloheximide (20 mg/ml) for different time periods. The protein levels of the Myc-hTDP-43 were analyzed by western blotting with anti-TDP-43 or anti-Myc antibody. The graph below the blots showed a quantification of the relative values at each time point, from which the half-lives of the proteins were estimated. Half-life was calculated by linear regression. Data were presented as the mean ± SEM ( n = 3) (*** p

    Article Snippet: Five-hundred micrograms of total protein were mixed with 2 μl anti-Znf179 or anti-TDP-43 antibodies (Proteintech Group Cat# 12892–1-AP RRID:AB_2200505) and added to the Antibody Capture Affinity Ligand in 1× Wash Buffer to make a final volume of 500 ul.

    Techniques: Solubility, Stable Transfection, Western Blot

    Knockout of Znf179 enhances TDP-43 aggregate formation in mice cortex and hippocampus. a and b The brain sections of wild-type ( a ) or Znf179-knockout mice ( b ) were stained with anti–TDP-43 antibody (green) and the nuclei were labeled with DAPI (blue). Scale bars = 100 μm. c and d The brain sections of wild-type ( c ) or Znf179-knockout mice ( d ) were stained with anti-TDP-43 antibody and the punctate staining of TDP-43 aggregates in the cortex region were indicated by white arrows. Scale bars = 100 μm. e and f The brain sections of wild-type ( e ) or Znf179-knockout mice ( f ) were stained with anti-TDP-43 antibody. Scale bars = 100 μm

    Journal: Journal of Biomedical Science

    Article Title: Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

    doi: 10.1186/s12929-018-0479-4

    Figure Lengend Snippet: Knockout of Znf179 enhances TDP-43 aggregate formation in mice cortex and hippocampus. a and b The brain sections of wild-type ( a ) or Znf179-knockout mice ( b ) were stained with anti–TDP-43 antibody (green) and the nuclei were labeled with DAPI (blue). Scale bars = 100 μm. c and d The brain sections of wild-type ( c ) or Znf179-knockout mice ( d ) were stained with anti-TDP-43 antibody and the punctate staining of TDP-43 aggregates in the cortex region were indicated by white arrows. Scale bars = 100 μm. e and f The brain sections of wild-type ( e ) or Znf179-knockout mice ( f ) were stained with anti-TDP-43 antibody. Scale bars = 100 μm

    Article Snippet: Five-hundred micrograms of total protein were mixed with 2 μl anti-Znf179 or anti-TDP-43 antibodies (Proteintech Group Cat# 12892–1-AP RRID:AB_2200505) and added to the Antibody Capture Affinity Ligand in 1× Wash Buffer to make a final volume of 500 ul.

    Techniques: Knock-Out, Mouse Assay, Staining, Labeling

    Znf179 interacts with TDP-43. a and b 293 T cells transiently transfected with Flag-mZnf179 ( a ) or N2a cells stably expressing GFP-mZnf179 ( b ) were immunoprecipitated with anti-TDP-43 or anti-Znf179 antibodies and immunoblotted with ant-TDP-43 and anti-Znf179 antibodies. c and d The total brain lysates of wild-type or Znf179-knockout mice were immunoprecipitated with either anti-TDP-43 or anti-Znf179 antibodies and immunoblotted with both anti-Znf179 and anti-TDP-43 antibodies

    Journal: Journal of Biomedical Science

    Article Title: Znf179 E3 ligase-mediated TDP-43 polyubiquitination is involved in TDP-43- ubiquitinated inclusions (UBI) (+)-related neurodegenerative pathology

    doi: 10.1186/s12929-018-0479-4

    Figure Lengend Snippet: Znf179 interacts with TDP-43. a and b 293 T cells transiently transfected with Flag-mZnf179 ( a ) or N2a cells stably expressing GFP-mZnf179 ( b ) were immunoprecipitated with anti-TDP-43 or anti-Znf179 antibodies and immunoblotted with ant-TDP-43 and anti-Znf179 antibodies. c and d The total brain lysates of wild-type or Znf179-knockout mice were immunoprecipitated with either anti-TDP-43 or anti-Znf179 antibodies and immunoblotted with both anti-Znf179 and anti-TDP-43 antibodies

    Article Snippet: Five-hundred micrograms of total protein were mixed with 2 μl anti-Znf179 or anti-TDP-43 antibodies (Proteintech Group Cat# 12892–1-AP RRID:AB_2200505) and added to the Antibody Capture Affinity Ligand in 1× Wash Buffer to make a final volume of 500 ul.

    Techniques: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Knock-Out, Mouse Assay

    Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to LC3‐II were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).

    Journal: Journal of Neurochemistry

    Article Title: Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1‐linked amyotrophic lateral sclerosis mice

    doi: 10.1111/jnc.14285

    Figure Lengend Snippet: Cystatin C (CysC) administration reduces insoluble p62 and poly‐ubiquitin accumulation in the lumbar spinal cords of early symptomatic SOD1 G93A mice. (a) Immunoblotting analyses of p62 and poly‐ubiquitin (poly‐Ubi). Note that detergent (1% Triton X‐100)‐insoluble p62 and poly‐Ubi was accumulated in SOD1 G93A mice, but they were not affected by CysC administration. (b) Co‐localization of p62 and poly‐Ubi aggregates were predominantly observed in outside of neurons. A representative image for the lumbar spinal cord section of the end‐stage SOD1 G93A mice with CysC administration stained for p62, poly‐ubiquitin, and CysC, along with a merged image with DAPI. (c) Immunoblotting analyses of spinal cords derived from the early symptomatic stage (4 months old) SOD1 G93A mice administered with phosphate‐buffered saline (PBS) or CysC for 2 weeks. In contrast to the end‐stage samples (a), the amounts of insoluble p62 and poly‐ubiqutinated proteins were reduced by CysC administration, suggesting the improved protein degradation. It should be also noted that bands corresponding to LC3‐II were hardly detected (arrowhead). (d) Astrocytes and microglia in the lumbar spinal cords of end‐stage SOD1 G93A mice, with or without administration of CysC, were immunostained with antibodies against glial fibrillary acidic protein (GFAP) and Iba‐1 with DAPI. Scale bars: 50 μm (b), and 100 μm (d).

    Article Snippet: Antibodies used were as follows: anti‐phosphorylated AMPK (Thr172) (1:250, #sc‐33524, RRID: AB_2169714, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐LC3 (1:500, #NB100‐2331, RRID: AB_10001955, Novus Biologicals LLC, Littleton, CO, USA), anti‐CysC (1 : 500 for immunoblotting, 1 : 100 for immunohistochemistry, #ab109508, RRID: AB_10888303, Abcam, Cambridge, UK), anti‐β‐actin (1 : 5000, #5441, RRID: AB_476744, Sigma‐Aldrich Co LLC, St. Louis, MO, USA), anti‐influenza hemagglutinin‐tag (HA‐tag) (1 : 500, #11‐867‐423‐001, RRID: AB_10094468, Roche, Basel, Switzerland), anti‐p62 (for immunoblotting, 1 : 1000, #PM045, RRID: AB_2169714, Medical and Biological Laboratories (MBL), Nagoya, Japan) (for immunohistochemistry, 1 : 100, #03‐GP62‐C, RRID: AB_1542690, American Research Products Inc., Belmont, MA, USA), anti‐poly‐ubiquitin (poly‐Ubi) (1 : 1000 for immunoblotting, 1:500 for immunohistochemistry, #D058‐3, RRID: AB_592937, MBL), anti‐glial fibrillary acidic protein (1 : 250, #G3893, RRID: AB_477010, Sigma‐Aldrich), and anti‐Iba‐1 (1:500, #019‐10741, Wako Pure Chemical Industries Ltd., Osaka, Japan).

    Techniques: Mouse Assay, Staining, Derivative Assay

    Cystatin C (CysC) activates the AMP‐activated kinase (AMPK) pathway to reduce the insoluble mutant SOD1 species. (a–e) Immunoblotting analyses of phosphorylated AMPK (pAMPK) (a and b), soluble or insoluble mutant SOD1 species (a and c), and LC3 (a, d and e) in the lumbar spinal cords of 5‐month‐old non‐transgenic control mice (nTg), phosphate‐buffered saline (PBS)‐administered SOD1 G93A mice (G93A PBS), and CysC‐administered SOD1 G93A mice (G93A CysC) at end‐stage. The level of LC3‐II was normalized by two distinct internal controls; LC3‐I (the pre‐modified form of LC3‐II; (d) or β‐actin (e). (f–h) The amount of SOD1 protein was negatively correlated with that of CysC in the ventral horn neurons. The lumbar spinal cord sections of end‐stage SOD1 G93A mice administered with CysC were immunostained for SOD1 and CysC (F and G). The fluorescent intensities were quantified and plotted for at least 50 ventral horn neurons of CysC‐infused SOD1 G93A mice (h). Representative fluorescence images used for quantification are shown (f and g). Similar staining was also obtained in three independent experiments. Scale bars: 50 μm (f) and 20 μm (g).

    Journal: Journal of Neurochemistry

    Article Title: Intracerebroventricular administration of Cystatin C ameliorates disease in SOD1‐linked amyotrophic lateral sclerosis mice

    doi: 10.1111/jnc.14285

    Figure Lengend Snippet: Cystatin C (CysC) activates the AMP‐activated kinase (AMPK) pathway to reduce the insoluble mutant SOD1 species. (a–e) Immunoblotting analyses of phosphorylated AMPK (pAMPK) (a and b), soluble or insoluble mutant SOD1 species (a and c), and LC3 (a, d and e) in the lumbar spinal cords of 5‐month‐old non‐transgenic control mice (nTg), phosphate‐buffered saline (PBS)‐administered SOD1 G93A mice (G93A PBS), and CysC‐administered SOD1 G93A mice (G93A CysC) at end‐stage. The level of LC3‐II was normalized by two distinct internal controls; LC3‐I (the pre‐modified form of LC3‐II; (d) or β‐actin (e). (f–h) The amount of SOD1 protein was negatively correlated with that of CysC in the ventral horn neurons. The lumbar spinal cord sections of end‐stage SOD1 G93A mice administered with CysC were immunostained for SOD1 and CysC (F and G). The fluorescent intensities were quantified and plotted for at least 50 ventral horn neurons of CysC‐infused SOD1 G93A mice (h). Representative fluorescence images used for quantification are shown (f and g). Similar staining was also obtained in three independent experiments. Scale bars: 50 μm (f) and 20 μm (g).

    Article Snippet: Antibodies used were as follows: anti‐phosphorylated AMPK (Thr172) (1:250, #sc‐33524, RRID: AB_2169714, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti‐LC3 (1:500, #NB100‐2331, RRID: AB_10001955, Novus Biologicals LLC, Littleton, CO, USA), anti‐CysC (1 : 500 for immunoblotting, 1 : 100 for immunohistochemistry, #ab109508, RRID: AB_10888303, Abcam, Cambridge, UK), anti‐β‐actin (1 : 5000, #5441, RRID: AB_476744, Sigma‐Aldrich Co LLC, St. Louis, MO, USA), anti‐influenza hemagglutinin‐tag (HA‐tag) (1 : 500, #11‐867‐423‐001, RRID: AB_10094468, Roche, Basel, Switzerland), anti‐p62 (for immunoblotting, 1 : 1000, #PM045, RRID: AB_2169714, Medical and Biological Laboratories (MBL), Nagoya, Japan) (for immunohistochemistry, 1 : 100, #03‐GP62‐C, RRID: AB_1542690, American Research Products Inc., Belmont, MA, USA), anti‐poly‐ubiquitin (poly‐Ubi) (1 : 1000 for immunoblotting, 1:500 for immunohistochemistry, #D058‐3, RRID: AB_592937, MBL), anti‐glial fibrillary acidic protein (1 : 250, #G3893, RRID: AB_477010, Sigma‐Aldrich), and anti‐Iba‐1 (1:500, #019‐10741, Wako Pure Chemical Industries Ltd., Osaka, Japan).

    Techniques: Mutagenesis, Transgenic Assay, Mouse Assay, Modification, Fluorescence, Staining

    Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), unc503 ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).

    Journal: eLife

    Article Title: PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis

    doi: 10.7554/eLife.33800

    Figure Lengend Snippet: Promoter restricted expression of human PAX3-FOXO1 has different effects on survival in developing zebrafish. Shown are the survival curves for the following promoters and the total number of zebrafish embryos analyzed for each condition: beta actin ( n = 120), mitfa ( n = 45), fli1 ( n = 81), ubi ( n = 160), unc503 ( n = 108), cmv ( n = 20), SA-splice acceptor ( n = 153).

    Article Snippet: The ubi promoter was a kind gift from Len Zon (Addgene #27320) , the unc503 promoter from Peter Currie (Addgene #64020) , the fli1 promoter and 3’ entry 2A-mCherry from Nathan Lawson (Addgene #31160 and #26031) ( ; ), and the mitfa promoter from James Lister (Addgene #81234).

    Techniques: Expressing

    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

    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

    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