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: The following antibodies were used for IP: GFP (AFP5002, Qbiogene), GFP (G1544, Sigma), and huntingtin (MAB5490, MilliporeSigma).

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: The following antibodies were used for IP: GFP (AFP5002, Qbiogene), GFP (G1544, Sigma), and huntingtin (MAB5490, MilliporeSigma).

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

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 ) Overexpression of mHtt reduces Nedd4 levels in rat primary cortical neurons. Cells were transduced with lentivirus expressing GFP or Htt Exon1-72Q, harvested on indicated days, and analyzed by Western blot. GFP, aggregated (agg) and soluble (sol) Htt were detected. n = 3, one-sample t test. ( B ) Filter trap-dot blotting analysis of Htt aggregation. Twenty-four hours after plating, mouse cortical neurons were transduced with lentivirus expressing either Htt Ex1-25Q or Htt Ex1-72Q and cultured for 6 days. Htt aggregates were detected by anti-EM48 antibody (MAB5374). Intensity of aggregated Htt was quantified ( n = 3, two-tailed Student’s t test, P = 0.0091) and presented as mean values ± SEM. ( C ) Nedd4 levels are reduced in brains of R6/2 model of HD. WT and R6/2 cerebra were homogenized and analyzed by Western blot using antibodies against EM48, Beclin 1 (Becn1), and p62. n = 3, two-tailed unpaired t test. ( D ) Nedd4 levels are reduced in brains of HD140Q KI mice. Cortical lysates of WT and HD140Q KI were analyzed by Western blot using antibodies against Nedd4 and Htt. Nedd4 values were normalized by Gapdh ( n = 6, two-tailed Student’s t test, P = 0.023). ( E ) mHtt overexpression in primary cortical neurons reduces phospho-S6 (p-S6) and Nedd4 levels. n = 3, one-sample t test. ( F ) Acute inhibition of mTORC1 activity by rapamycin reduces Nedd4 level in rat primary cortical neurons. Cells were treated with vehicle (veh) or 20 nM rapamycin (rapa) for 24 hours on DIV9, harvested, and analyzed by Western blot using antibodies against Nedd4, p-S6, and S6. n = 3, two-tailed unpaired t test. β-actin (actin), Gapdh and Ponceau S (Pon S) were used as loading controls. * P < 0.05; ** P < 0.01; NS, not significant.

Article Snippet: The following antibodies were used for IP: GFP (AFP5002, Qbiogene), GFP (G1544, Sigma), and huntingtin (MAB5490, MilliporeSigma).

Techniques: Over Expression, Transduction, Expressing, Western Blot, Cell Culture, Two Tailed Test, Inhibition, Activity Assay