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

Journal: Cell Biology and Toxicology

Article Title: METTL7B-stabilized lncRNA-MIR22HG to drive p53-mediated neuronal apoptosis via the ubiquitinating JARID2 in cerebral ischemia/reperfusion injury

doi: 10.1007/s10565-026-10157-4

Figure Lengend Snippet: Temporal expression and subcellular localization of lncRNA-MIR22HG after CIR. A , B qRT-PCR: Relative lncRNA-MIR22HG expression in mouse cortices following MCAO/R ( A ) and in N2a Cells after OGD/R ( B ) at the indicated time points. n = 3 per group. C FISH images: Subcellular localization of lncRNA-MIR22HG (red) in N2a Cells at different OGD/R time points. DAPI-stained Nuclei (blue). Scale bar, 50 μm. D Nuclear-to-cytoplasmic ratio of MIR22HG signals at pre-determined times. ( n = 6/group). * P < 0.05, ** P < 0.01 vs. control

Article Snippet: Seeking the assessment of the impact of in vitro IRI, we grew mouse neuroblastoma N2a cells (ATCC, CCL-131).

Techniques: Expressing, Quantitative RT-PCR, Staining, Control

Journal: Cell Biology and Toxicology

Article Title: METTL7B-stabilized lncRNA-MIR22HG to drive p53-mediated neuronal apoptosis via the ubiquitinating JARID2 in cerebral ischemia/reperfusion injury

doi: 10.1007/s10565-026-10157-4

Figure Lengend Snippet: Impacts of lncRNA-MIR22HG on neuronal viability and apoptosis following OGD/R. A qRT-PCR: Relative expression of MIR22HG in N2a Cells subjected to transfection with si-NC, si-MIR22HG, pcDNA3.1-NC, or pcDNA3.1-MIR22HG. B CCK-8 assay: Cell viability in different groups. C LDH release as an indicator of cell membrane integrity. D Flow cytometry plots for Annexin V-FITC/PI staining: Apoptosis in each group. E Statistical analysis of apoptotic cell percentages. ( n = 6/group). ** P < 0.01, *** P < 0.001 vs. control or si-NC; # P < 0.05, ## P < 0.01 vs. pcDNA3.1-NC; && P < 0.01 vs. si-NC

Article Snippet: Seeking the assessment of the impact of in vitro IRI, we grew mouse neuroblastoma N2a cells (ATCC, CCL-131).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Membrane, Flow Cytometry, Staining, Control

Journal: Cell Biology and Toxicology

Article Title: METTL7B-stabilized lncRNA-MIR22HG to drive p53-mediated neuronal apoptosis via the ubiquitinating JARID2 in cerebral ischemia/reperfusion injury

doi: 10.1007/s10565-026-10157-4

Figure Lengend Snippet: MIR22HG activates MDM2/p300/p53 apoptotic signaling in OGD/R-subjected N2a Cells. A , C WB: expression of MDM2, p300, Bax, Puma, and Noxa in N2a Cells after knockdown ( A ) or overexpression ( C ) of MIR22HG. Loading control (β-actin). B , D Relative protein expression levels. E qRT-PCR: mRNA levels of the indicated apoptotic genes. ( n = 3/group). ** P < 0.01 vs. control or si-NC; # P < 0.05, ## P < 0.01 vs. pcDNA3.1-NC

Article Snippet: Seeking the assessment of the impact of in vitro IRI, we grew mouse neuroblastoma N2a cells (ATCC, CCL-131).

Techniques: Expressing, Knockdown, Over Expression, Control, Quantitative RT-PCR

Journal: Cell Biology and Toxicology

Article Title: METTL7B-stabilized lncRNA-MIR22HG to drive p53-mediated neuronal apoptosis via the ubiquitinating JARID2 in cerebral ischemia/reperfusion injury

doi: 10.1007/s10565-026-10157-4

Figure Lengend Snippet: lncRNA-MIR22HG stabilizes JARID2 protein by inhibiting its ubiquitin-mediated degradation. A RIP-qPCR: Enrichment of MIR22HG in JARID2 immunoprecipitates compared to IgG control. B qRT-PCR: Relative JARID2 mRNA expression after MIR22HG knockdown or overexpression. C , D WB and quantification of JARID2 protein levels in N2a Cells transfected with si-MIR22HG or oe-MIR22HG. D , E , F Proteasome inhibitor (MG132) and cycloheximide (CHX) chase assays: MIR22HG knockdown accelerates JARID2 degradation, whereas its overexpression prolongs JARID2 half-life. J Ubiquitination assay: Decreased K48-linked ubiquitination of JARID2 in MIR22HG-overexpressing cells and increased ubiquitination after MIR22HG silencing. H Co-immunoprecipitation: JARID2 knockdown impairs the interaction of p53 with its cofactors (p300 and MDM2). ( n = 3); ** P < 0.01, *** P < 0.001 vs. controls

Article Snippet: Seeking the assessment of the impact of in vitro IRI, we grew mouse neuroblastoma N2a cells (ATCC, CCL-131).

Techniques: Ubiquitin Proteomics, Control, Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Transfection, Immunoprecipitation

Journal: Cell Biology and Toxicology

Article Title: METTL7B-stabilized lncRNA-MIR22HG to drive p53-mediated neuronal apoptosis via the ubiquitinating JARID2 in cerebral ischemia/reperfusion injury

doi: 10.1007/s10565-026-10157-4

Figure Lengend Snippet: METTL7B is up-regulated after CIRI. A , B qRT-PCR: Time-dependent induction of METTL7B mRNA in MCAO mouse cortices ( A ) and OGD-challenged N2a Cells ( B ). C , D WB and quantification of METTL7B protein expression in vivo and in vitro at the pre-determined reperfusion times. ( n = 3); * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control

Article Snippet: Seeking the assessment of the impact of in vitro IRI, we grew mouse neuroblastoma N2a cells (ATCC, CCL-131).

Techniques: Quantitative RT-PCR, Expressing, In Vivo, In Vitro, Control