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ATCC n2a mouse neuroblastoma cells

Endogenous Grx1 is upregulated in <t>N2a-hTDP-43</t> cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; <t>N2a-hTDP-43,</t> <t>neuro-2a</t> cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

N2a Mouse Neuroblastoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 4098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation"

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

Journal: Neuroreport

doi: 10.1097/WNR.0000000000002266

Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Figure Legend Snippet: Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Techniques Used: Biomarker Discovery, Over Expression, Transfection, Control, Staining, Expressing, Binding Assay

Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Figure Legend Snippet: Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Techniques Used: Biomarker Discovery, Over Expression, Transfection, Staining, Control, Expressing, Binding Assay

$Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.$
Figure Legend Snippet: Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.

Techniques Used: Staining, Transfection, Lysis, Western Blot, Expressing, Binding Assay

Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Figure Legend Snippet: Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Techniques Used: Transfection, Staining, Binding Assay

Image Search Results

Journal: Neuroreport

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: N2a mouse neuroblastoma cells (CCL-131, ATCC, Manassas, Virginia, USA) were plated at a density of 2 × 10 5 cells/ml and grown in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C, in a humidified 5% CO 2 incubator.

Techniques: Biomarker Discovery, Over Expression, Transfection, Control, Staining, Expressing, Binding Assay

Journal: Neuroreport

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Techniques: Biomarker Discovery, Over Expression, Transfection, Staining, Control, Expressing, Binding Assay

Journal: Neuroreport

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.

Techniques: Staining, Transfection, Lysis, Western Blot, Expressing, Binding Assay

Journal: Neuroreport

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Techniques: Transfection, Staining, Binding Assay

N2a cells were treated with CI-994, W2A-28, W2A-42 and W2A-43 at 8 μM for 24 h, and the level of β-catenin was examined by Western blotting. Data represent mean ± SEM from four independent experiments. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test.

Journal: bioRxiv

Article Title: Discovery of a CI-994 derivative as a dual modulator of class I HDACs and Wnt/β-catenin signaling for Alzheimer’s disease therapy

doi: 10.64898/2026.04.30.721954

Figure Lengend Snippet: N2a cells were treated with CI-994, W2A-28, W2A-42 and W2A-43 at 8 μM for 24 h, and the level of β-catenin was examined by Western blotting. Data represent mean ± SEM from four independent experiments. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: HEK293 cells (CRL-1573) and N2a neuroblastoma cells (CCL-131) were obtained from ATCC.

Techniques: Western Blot, Comparison

AS-IV attenuates brain oxidative stress and inflammatory signaling in DSS-induced colitis and protects against LPS-induced neuronal injury in vitro. ( A ) Representative H&E-stained brain sections from CON, DSS, and DSS + AS-IV (100 mg/kg) groups (2.5× and 10×; scale bar = 100 μm, n = 6). ( B ) Western blot quantification of brain inflammatory and oxidative/nitrosative stress markers (iNOS, 3-nitrotyrosine, CYP2E1, COX-2); GAPDH as loading control ( n = 3 technical replicates from pooled lysates). ( C ) Western blot quantification of phosphorylated MAPK and NF-κB pathway proteins (p-ERK, p-JNK, p-p38, p-NF-κB, p-IκBα) in brain tissue ( n = 3 technical replicates from pooled lysates). Representative immunofluorescence images of DCF-DA ( D ) and cleaved caspase-3 ( E ) in Neuro2a cells treated with CON, LPS (100 μg/mL), or LPS + AS-IV (5 μM); nuclei stained with DAPI (blue) ( n = 6). ( F ) Quantification of intracellular ROS levels through DCF-DA assay. ( G ) Cell viability assessed through CCK-8 assay. Data are presented as means ± SEM. ** p < 0.01, *** p < 0.005 vs. CON group; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. DSS or LPS group.

Journal: Antioxidants

Article Title: Astragaloside IV from Astragalus membranaceus Fisch. ex Bunge Mitigates DSS-Induced Colitis via Anti-Inflammatory and Antioxidant Modulation of the Gut–Liver–Brain Axis

doi: 10.3390/antiox15040474

Figure Lengend Snippet: AS-IV attenuates brain oxidative stress and inflammatory signaling in DSS-induced colitis and protects against LPS-induced neuronal injury in vitro. ( A ) Representative H&E-stained brain sections from CON, DSS, and DSS + AS-IV (100 mg/kg) groups (2.5× and 10×; scale bar = 100 μm, n = 6). ( B ) Western blot quantification of brain inflammatory and oxidative/nitrosative stress markers (iNOS, 3-nitrotyrosine, CYP2E1, COX-2); GAPDH as loading control ( n = 3 technical replicates from pooled lysates). ( C ) Western blot quantification of phosphorylated MAPK and NF-κB pathway proteins (p-ERK, p-JNK, p-p38, p-NF-κB, p-IκBα) in brain tissue ( n = 3 technical replicates from pooled lysates). Representative immunofluorescence images of DCF-DA ( D ) and cleaved caspase-3 ( E ) in Neuro2a cells treated with CON, LPS (100 μg/mL), or LPS + AS-IV (5 μM); nuclei stained with DAPI (blue) ( n = 6). ( F ) Quantification of intracellular ROS levels through DCF-DA assay. ( G ) Cell viability assessed through CCK-8 assay. Data are presented as means ± SEM. ** p < 0.01, *** p < 0.005 vs. CON group; # p < 0.05, ## p < 0.01, ### p < 0.005 vs. DSS or LPS group.

Article Snippet: N2a neuroblastoma cells (CCL-131, ATCC, Manassas, VA, USA), T84 human intestinal epithelial cells, and AML12 murine hepatocytes were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin–streptomycin.

Techniques: In Vitro, Staining, Western Blot, Control, Immunofluorescence, CCK-8 Assay

Cytotoxicity and cell viability in mistranslating human and murine cells. Plasmids encoding mCherry and wild-type human tRNA Ser AGA or variant tRNA Ser AAA with different flanking sequence contexts from human tRNA Ser AGA 2–2, 2–3, 2–4, and 2–5 genes were transfected into ( A ) human HEK 293T and ( B ) murine N2a cells; ( A, B ) cytotoxicity was determined at 24 h post-transfection using CytotoxGlo. ( C, D ) Cell viability (CCK8 assay) was also measured in ( C ) HEK 293T cells and in ( D ) N2a cells at 24 h after transfection with a plasmid expressing the wildtype GFP-mCherry reporter and the indicated wildtype tRNA Ser AGA or AAA anticodon mutant. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on at least N = 3 biological replicates (n.s., not significant; * P <.05).

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Cytotoxicity and cell viability in mistranslating human and murine cells. Plasmids encoding mCherry and wild-type human tRNA Ser AGA or variant tRNA Ser AAA with different flanking sequence contexts from human tRNA Ser AGA 2–2, 2–3, 2–4, and 2–5 genes were transfected into ( A ) human HEK 293T and ( B ) murine N2a cells; ( A, B ) cytotoxicity was determined at 24 h post-transfection using CytotoxGlo. ( C, D ) Cell viability (CCK8 assay) was also measured in ( C ) HEK 293T cells and in ( D ) N2a cells at 24 h after transfection with a plasmid expressing the wildtype GFP-mCherry reporter and the indicated wildtype tRNA Ser AGA or AAA anticodon mutant. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on at least N = 3 biological replicates (n.s., not significant; * P <.05).

Article Snippet: Mammalian cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States), and cell culture work was conducted in the human embryonic kidney (HEK) 293T cells (CRL-3216, ATCC), in murine neuroblastoma Neuro-2a (N2a) cells (CCL-131, ATCC), or in human K562 lymphoblast cells (CCL-243, ATCC) as indicated.

Techniques: Variant Assay, Sequencing, Transfection, CCK-8 Assay, Plasmid Preparation, Expressing, Mutagenesis, Standard Deviation

Quantifying mistranslation levels in live human and murine cells by flow cytometry. Flow-cytometry was used to measure GFP and mCherry fluorescence in 2.5 × 10 6 individual viable HEK 293T (above) or murine N2a cells (below) transfected with the wildtype GFP-mCherry or GFP-mCherry S151F mutant and the indicated wildtype tRNA Ser AGA or tRNA Ser AAA variant. ( A ) The fluorescence measurements were converted into heatmaps to depict the population of cells observed across mCherry and GFP fluorescence levels. ( B ) To determine the mistranslation level resulting from each tRNA mutant in the indicated cell lines, the mCherry/GFP fluorescence ratio from individual cells were calculated and plotted. The data were normalized to the wildtype mCherry/GFP fluorescence ratio observed in mistranslating cells. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on N = 3 biological replicates (n.s., not significant; ** P <.01; *** P <.001; **** P <.0001).

Journal: Nucleic Acids Research

Article Title: Mistranslation from an endogenous tRNA variant in human pan-genome cell lines

doi: 10.1093/nar/gkag224

Figure Lengend Snippet: Quantifying mistranslation levels in live human and murine cells by flow cytometry. Flow-cytometry was used to measure GFP and mCherry fluorescence in 2.5 × 10 6 individual viable HEK 293T (above) or murine N2a cells (below) transfected with the wildtype GFP-mCherry or GFP-mCherry S151F mutant and the indicated wildtype tRNA Ser AGA or tRNA Ser AAA variant. ( A ) The fluorescence measurements were converted into heatmaps to depict the population of cells observed across mCherry and GFP fluorescence levels. ( B ) To determine the mistranslation level resulting from each tRNA mutant in the indicated cell lines, the mCherry/GFP fluorescence ratio from individual cells were calculated and plotted. The data were normalized to the wildtype mCherry/GFP fluorescence ratio observed in mistranslating cells. Error bars represent ±1 standard deviation of the mean and statistical analysis was calculated using single factor ANOVA and was based on N = 3 biological replicates (n.s., not significant; ** P <.01; *** P <.001; **** P <.0001).

Techniques: Flow Cytometry, Fluorescence, Transfection, Mutagenesis, Variant Assay, Standard Deviation

( A ) Interaction between Nedd4 and Htt Exon1-GFP. 293FT cells were transfected with Nedd4 together with GFP, Htt Exon1-25Q-GFP, or Htt Exon1-46Q-GFP. Cells were harvested 48 hours after transfection, and co-IP was performed followed by Western blot analysis. ( B ) Interaction of Nedd4 with Htt480-68Q. N2a cells were transfected with Nedd4 and Htt480-68Q and collected 26 hours after transfection. Co-IP was performed followed by Western blot analysis. T7 antibody was used to detect T7-tagged Nedd4. ( C ) Immunoblot analysis of Nedd4 expression in mouse brain regions: cortex (Ctx), cerebellum (Cer), striatum (Str), and hippocampus (Hip). Nedd4 was detected using an anti-Nedd4 antibody (Proteintech, 21698-1-AP), and Gapdh served as a loading control. ( D ) Quantification of relative Nedd4 expression normalized to Gapdh ( n = 7; one-way ANOVA with Tukey’s post hoc test; Cer vs. Str, P = 0.0461). ( E ) Endogenous interaction between Htt and Nedd4 in mouse cortex. Co-IP was performed using cortical lysates, with Htt immunoprecipitated using an Htt antibody (EPR5526). Nedd4 was detected in the pulldown. IgG served as a negative control. ( F ) Nedd4 promotes Htt ubiquitination in a ligase activity–dependent manner. N2a cells were transfected with Htt571-72Q and HA-ubiquitin together with vector control, Nedd4, or catalytically inactive Nedd4-CS. Cells were harvested 50 hours after transfection, and denaturing IP was followed by Western blotting. n = 3; one-way ANOVA with Tukey’s multiple-comparison test (* P < 0.01; ** P < 0.001; NS, not significant). ( G ) Htt undergoes monoubiquitination at multiple lysine residues. N2a cells were transfected with GFP-Htt480-68Q and either WT HA-ubiquitin or lysine-free HA-K0 ubiquitin. Denaturing IP and Western blot analysis were performed 25 hours after transfection. N4, Nedd4; Ub, ubiquitin; vec, vector; CS, Nedd4 CS; Pon S, Ponceau S.

Journal: JCI Insight

Article Title: Ubiquitin ligase Nedd4 regulates the abundance and toxicity of mutant huntingtin

doi: 10.1172/jci.insight.181013

Figure Lengend Snippet: ( A ) Interaction between Nedd4 and Htt Exon1-GFP. 293FT cells were transfected with Nedd4 together with GFP, Htt Exon1-25Q-GFP, or Htt Exon1-46Q-GFP. Cells were harvested 48 hours after transfection, and co-IP was performed followed by Western blot analysis. ( B ) Interaction of Nedd4 with Htt480-68Q. N2a cells were transfected with Nedd4 and Htt480-68Q and collected 26 hours after transfection. Co-IP was performed followed by Western blot analysis. T7 antibody was used to detect T7-tagged Nedd4. ( C ) Immunoblot analysis of Nedd4 expression in mouse brain regions: cortex (Ctx), cerebellum (Cer), striatum (Str), and hippocampus (Hip). Nedd4 was detected using an anti-Nedd4 antibody (Proteintech, 21698-1-AP), and Gapdh served as a loading control. ( D ) Quantification of relative Nedd4 expression normalized to Gapdh ( n = 7; one-way ANOVA with Tukey’s post hoc test; Cer vs. Str, P = 0.0461). ( E ) Endogenous interaction between Htt and Nedd4 in mouse cortex. Co-IP was performed using cortical lysates, with Htt immunoprecipitated using an Htt antibody (EPR5526). Nedd4 was detected in the pulldown. IgG served as a negative control. ( F ) Nedd4 promotes Htt ubiquitination in a ligase activity–dependent manner. N2a cells were transfected with Htt571-72Q and HA-ubiquitin together with vector control, Nedd4, or catalytically inactive Nedd4-CS. Cells were harvested 50 hours after transfection, and denaturing IP was followed by Western blotting. n = 3; one-way ANOVA with Tukey’s multiple-comparison test (* P < 0.01; ** P < 0.001; NS, not significant). ( G ) Htt undergoes monoubiquitination at multiple lysine residues. N2a cells were transfected with GFP-Htt480-68Q and either WT HA-ubiquitin or lysine-free HA-K0 ubiquitin. Denaturing IP and Western blot analysis were performed 25 hours after transfection. N4, Nedd4; Ub, ubiquitin; vec, vector; CS, Nedd4 CS; Pon S, Ponceau S.

Article Snippet: HEK-293FT (Invitrogen R70007 ) and murine neuroblastoma cell line N2A (ATCC CCL-131) were cultured in DMEM with 10% FBS.

Techniques: Transfection, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Immunoprecipitation, Negative Control, Ubiquitin Proteomics, Activity Assay, Plasmid Preparation, Comparison

( A ) Htt levels are reduced by overexpression of Nedd4 in a ligase activity–dependent manner in N2a cells. Cells were transfected with Htt571-72Q together with vector (vec), Nedd4 (N4), or Nedd4 CS (CS), and analyzed by Western blot 2 and 3 days after transfection. n = 4, one-way ANOVA with Tukey’s multiple-comparison test. ( B ) Htt degradation is enhanced by overexpression of Nedd4 in a ligase activity–dependent manner in N2a cells. Cells were transfected with Htt571-72Q together with vector, Nedd4, or Nedd4 CS. Cycloheximide (CHX) 6-hour-chase experiment was followed by Western blot. n = 3, two-way ANOVA with Tukey’s multiple-comparison test. ( C ) Nedd4 knockdown increases Htt levels in N2a cells. Cells were cotransfected with Htt571-72Q and scrambled (scr) or shNedd4-35 (N4-35) plasmid, harvested at the indicated time points, and analyzed by Western blot. n = 3, one-sample t test. ( D ) Nedd4 knockdown impairs Htt degradation in N2a cells. Cells were transfected with scr or N4-35 plasmid. Forty-eight hours later, they were transfected with Htt571-72Q, subjected to 6-, 12-, and 24-hour CHX-chase experiment, harvested and analyzed by Western blot. n = 3, two-way ANOVA with Šídák’s multiple-comparison test. ( E ) Nedd4 knockdown increases Htt levels in mouse primary cortical neurons. Cells were transduced with lentivirus expressing Htt571-72Q together with scrambled (scr), shNedd4-34 (N4-34), or N4-35 lentivirus, harvested, and analyzed by Western blot. n = 3, one-way ANOVA with Dunnett’s multiple-comparison test. ( F ) Nedd4 knockdown increases endogenous Htt levels in mouse primary cortical neurons. Twenty-four hours after plating, cells were transduced with lentivirus expressing GFP and either scr or N4-35, cultured for an additional 6 days, and immunostained with anti-Htt antibody (5656S). Htt intensity was quantified in double-transduced cells ( n = 26 fields, 2-tailed Student t test; scale bar: 20 μm). α-tubulin (α-tub), loading control. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant.

Journal: JCI Insight

Article Title: Ubiquitin ligase Nedd4 regulates the abundance and toxicity of mutant huntingtin

doi: 10.1172/jci.insight.181013

Figure Lengend Snippet: ( A ) Htt levels are reduced by overexpression of Nedd4 in a ligase activity–dependent manner in N2a cells. Cells were transfected with Htt571-72Q together with vector (vec), Nedd4 (N4), or Nedd4 CS (CS), and analyzed by Western blot 2 and 3 days after transfection. n = 4, one-way ANOVA with Tukey’s multiple-comparison test. ( B ) Htt degradation is enhanced by overexpression of Nedd4 in a ligase activity–dependent manner in N2a cells. Cells were transfected with Htt571-72Q together with vector, Nedd4, or Nedd4 CS. Cycloheximide (CHX) 6-hour-chase experiment was followed by Western blot. n = 3, two-way ANOVA with Tukey’s multiple-comparison test. ( C ) Nedd4 knockdown increases Htt levels in N2a cells. Cells were cotransfected with Htt571-72Q and scrambled (scr) or shNedd4-35 (N4-35) plasmid, harvested at the indicated time points, and analyzed by Western blot. n = 3, one-sample t test. ( D ) Nedd4 knockdown impairs Htt degradation in N2a cells. Cells were transfected with scr or N4-35 plasmid. Forty-eight hours later, they were transfected with Htt571-72Q, subjected to 6-, 12-, and 24-hour CHX-chase experiment, harvested and analyzed by Western blot. n = 3, two-way ANOVA with Šídák’s multiple-comparison test. ( E ) Nedd4 knockdown increases Htt levels in mouse primary cortical neurons. Cells were transduced with lentivirus expressing Htt571-72Q together with scrambled (scr), shNedd4-34 (N4-34), or N4-35 lentivirus, harvested, and analyzed by Western blot. n = 3, one-way ANOVA with Dunnett’s multiple-comparison test. ( F ) Nedd4 knockdown increases endogenous Htt levels in mouse primary cortical neurons. Twenty-four hours after plating, cells were transduced with lentivirus expressing GFP and either scr or N4-35, cultured for an additional 6 days, and immunostained with anti-Htt antibody (5656S). Htt intensity was quantified in double-transduced cells ( n = 26 fields, 2-tailed Student t test; scale bar: 20 μm). α-tubulin (α-tub), loading control. * P < 0.05; ** P < 0.01; *** P < 0.001; NS, not significant.

Article Snippet: HEK-293FT (Invitrogen R70007 ) and murine neuroblastoma cell line N2A (ATCC CCL-131) were cultured in DMEM with 10% FBS.

Techniques: Over Expression, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Comparison, Knockdown, Transduction, Expressing, Cell Culture, Control

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