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

Journal: Alzheimer's & Dementia

Article Title: NRN1 as a therapeutic target for Alzheimer's disease

doi: 10.1002/alz.71149

Figure Lengend Snippet: Abcam ab64186 recognizes NRN1 protein in mouse neuroblastoma cells. (A) Representative Western blot of human embryonic kidney (HEK) 293T, Neuro‐2a (N2a) mouse neuroblastoma, and SH‐SY5Y human neuroblastoma cell lysates (50 µg protein), probed with NRN1 polyclonal antibody Abcam ab64186 and GAPDH monoclonal antibody. (B) N2a cells were transfected with NRN1 or Scramble (non‐targeting) siRNA smart pools and harvested after 96 h. Western blot analyses with NRN1 polyclonal antibody Abcam ab64186 revealed reduced intensity of the ∼34 kDa band in NRN1‐depleted cells, compared to Scramble control. 10 µg of protein was loaded per lane, and GAPDH was probed as a loading control. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; NRN1, Neuritin‐1; siRNA, small interfering RNA;

Article Snippet: Neuro‐2a (N2a) mouse neuroblastoma cell lines (Catalog No.: CCL‐131, ATCC) were maintained in MEM (Catalog No.: 11095‐080, Thermo Fisher Scientific) with 10% FBS and 1% penicillin‐streptomycin.

Techniques: Western Blot, Transfection, Control, Small Interfering RNA