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ATCC murine neuroblastoma cell line n2a

( 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. <t>N2a</t> 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.

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1) Product Images from "Ubiquitin ligase Nedd4 regulates the abundance and toxicity of mutant huntingtin"

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

Journal: JCI Insight

doi: 10.1172/jci.insight.181013

Figure Legend 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.

Techniques Used: 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.

Figure Legend 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.

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

Cell Culture:

Article Title: Treatment with Efavirenz extends survival in Creutzfeldt-Jakob disease model by regulating brain cholesterol metabolism.
Article Snippet: .. Cell culture, transfection, and cell lysis The murine neuroblastoma cell line N2a was obtained from ATCC (CCL-131). ..

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Article Title: Ubiquitin ligase Nedd4 regulates the abundance and toxicity of mutant huntingtin.
Article Snippet: pLP/VSV-G was obtained from Invitrogen.

Article Title: Ubiquitin ligase Nedd4 regulates the abundance and toxicity of mutant huntingtin
Article Snippet: EGFP insert was amplified by PCR from pEGFP-C1 (Clontech) and was subcloned into the lentiviral expression vector pER4 at NheI/NotI sites.

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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

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

Figure 1. Effects of PSO on APP and Aβ42 levels in N2a cells challenged with LPS. (a) Representative Western blot images showing the levels of APP and Aβ42 in N2a cells treated with 1 µg/mL LPS in the absence or presence of PSO at varying concentrations (0.2, 10, and 25 µg/mL) (see Supplementary Material Section S1.1). (b) Quantification of APP levels normalized to β-actin. (c) Aβ42 levels normalized to β-actin. Data are presented as mean ± SD from three independent experiments. Statistical significance: * p < 0.05; ** p < 0.01; **** p < 0.0001, with asterisk (*) indicating the comparison versus control group; # p < 0.05; #### p < 0.0001, with (#) indicating the comparison with the LPS- treated group.

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients.

doi: 10.3390/biology14050548

Figure Lengend Snippet: Figure 1. Effects of PSO on APP and Aβ42 levels in N2a cells challenged with LPS. (a) Representative Western blot images showing the levels of APP and Aβ42 in N2a cells treated with 1 µg/mL LPS in the absence or presence of PSO at varying concentrations (0.2, 10, and 25 µg/mL) (see Supplementary Material Section S1.1). (b) Quantification of APP levels normalized to β-actin. (c) Aβ42 levels normalized to β-actin. Data are presented as mean ± SD from three independent experiments. Statistical significance: * p < 0.05; ** p < 0.01; **** p < 0.0001, with asterisk (*) indicating the comparison versus control group; # p < 0.05; #### p < 0.0001, with (#) indicating the comparison with the LPS- treated group.

Article Snippet: The murine neuroblastoma cell line Neuro-2a (N2a) (ATCC: CCL-131TM) was chosen as a neuronal cell model. N2a cells were kindly provided by Prof. Gaetano Donofrio from the Department of Medical-Veterinary Science of the University of Parma, Italy.

Techniques: Western Blot, Comparison, Control

Figure 2. The effects of PSO on LPS-induced tau and phosphorylated tau (p-tau181) levels in N2a cells. (a,c) Representative Western blot images showing the expression levels of tau and p-tau181 in N2a cell (see Supplementary Material Section S1.1). (b) Quantification of tau protein levels normalized to β-actin. (d) Quantification of p-tau181 levels normalized to β-actin. Statistical significance: * p < 0.05; ** p < 0.01, with asterisk (*) indicating the comparison versus control group; # p < 0.05, ## p < 0.01, ### p < 0.001, with (#) indicating the comparison with the LPS-treated group. Data are presented as the mean ± SD.

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients.

doi: 10.3390/biology14050548

Figure Lengend Snippet: Figure 2. The effects of PSO on LPS-induced tau and phosphorylated tau (p-tau181) levels in N2a cells. (a,c) Representative Western blot images showing the expression levels of tau and p-tau181 in N2a cell (see Supplementary Material Section S1.1). (b) Quantification of tau protein levels normalized to β-actin. (d) Quantification of p-tau181 levels normalized to β-actin. Statistical significance: * p < 0.05; ** p < 0.01, with asterisk (*) indicating the comparison versus control group; # p < 0.05, ## p < 0.01, ### p < 0.001, with (#) indicating the comparison with the LPS-treated group. Data are presented as the mean ± SD.

Techniques: Western Blot, Expressing, Comparison, Control

Figure 3. The effects of PSO on LPS-induced expression of inflammatory and oxidative stress markers in N2a cells treated with PSO. (a) Representative Western blot images showing the expression levels of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Groups include Control, LPS (1 µg/ mL), LPS co-treatment with different concentrations of PSO (0.2, 10, and 25 µg/mL), and PSO (0.2, 10, and 25 µg/mL) (see Supplementary Material Section S1.1). (b) Quantification of total iNOS protein levels normalized to β-actin. (c) Quantification of IL-1β protein levels normalized to β-actin. (d) Quantification of TNF-α protein levels normalized to β-actin. Statistical significance: * p < 0.05; ** p < 0.01; **** p < 0.0001, with asterisk (*) indicating the comparison versus control group; # p < 0.05, ## p < 0.01, with (#) indicating the comparison with the LPS-treated group. Data are presented as the mean ± SD.

Journal: Biology

Article Title: Neuroprotective, Antioxidant and Anti-Inflammatory Effect of Greek Pomegranate Seed Oil on N2a Neuroblastoma Cells and Mild Cognitive Impairment Patients.

doi: 10.3390/biology14050548

Figure Lengend Snippet: Figure 3. The effects of PSO on LPS-induced expression of inflammatory and oxidative stress markers in N2a cells treated with PSO. (a) Representative Western blot images showing the expression levels of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-alpha (TNF-α). Groups include Control, LPS (1 µg/ mL), LPS co-treatment with different concentrations of PSO (0.2, 10, and 25 µg/mL), and PSO (0.2, 10, and 25 µg/mL) (see Supplementary Material Section S1.1). (b) Quantification of total iNOS protein levels normalized to β-actin. (c) Quantification of IL-1β protein levels normalized to β-actin. (d) Quantification of TNF-α protein levels normalized to β-actin. Statistical significance: * p < 0.05; ** p < 0.01; **** p < 0.0001, with asterisk (*) indicating the comparison versus control group; # p < 0.05, ## p < 0.01, with (#) indicating the comparison with the LPS-treated group. Data are presented as the mean ± SD.

Techniques: Expressing, Western Blot, Control, Comparison

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murine neuroblastoma cell line n2a (ATCC)

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