mouse neuroblastoma n2a n2a cells  (ATCC)

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Genomic distribution of SP9 ChIP-seq peaks based on data from ( ; ). Peaks localize predominantly to enhancer-like regions (77%), with smaller fractions at promoters (6.2%) and other regions (16.8%). B , Motif enrichment within SP9 peaks. Promoter-associated peaks (left) are enriched for GC-rich motifs recognized by KLF and SP factors, whereas enhancer-like peaks (right) are enriched for TAATT motifs recognized by DLX homeobox factors. Within each block: motif logos (left), − log 10 𝑃 -value and target/background percentage of peaks containing the motif (middle), and log 2 enrichment (right). C , Schematic of the motif scan-based classification of SP9 peaks. Peaks were grouped based on the presence of an SP motif only (direct binding), a DLX motif only (indirect binding via DLX), or both (cobinding). D , Coarse genomic annotation (enhancer, transcription start site (TSS), or other) of the four SP9 peak categories defined in (C): SP9+DLX (both motifs present), DLX (DLX motif only), Other, and SP9 (SP motif only). E , Fine genomic annotation (exon, intron, intergenic, promoter, UTR, other) of the same four peak categories. F , Intersection of DLX2 and SP9 ChIP-seq peaks from E13.5 mouse GE, based on data from ( ; ; ). Bar plot shows the number of overlapping and non-overlapping peaks per category (DLX2 only, overlap, SP9 only). G , Whole-mount X-Gal staining (blue) of E11.5 transgenic mouse embryos carrying VISTA enhancer reporters hs119 (near Arx ), hs170 (near Fign ), hs883 (near Sox6 ), and hs298 (near Dlx6os1 ) used in the luciferase assays in panels K-N and shown as genome browser tracks in panel H. Images from . H , Genome browser tracks showing ChIP-seq signal for SP9, DLX2, and DLX5 in E13.5 mouse GE at the Sp9 and Six3 promoters (highlighted in grey) and the VISTA enhancers hs119, hs170, hs883, and hs298 (highlighted in blue). Numbers below each track indicate the count of SP and DLX motifs within the selected regulatory element. Data from ( ; ; ). I-N , Luciferase reporter activity in N2A cells transfected with combinations of Sp9 and Dlx2 , Dlx5 , or Dlx6 as indicated, driven by the Sp9 promoter ( I ), Six3 promoter ( J ), or enhancer regions hs119 of Arx ( K ), hs170 of Fign ( L ), hs883 of Sox6 ( M ), and hs298 of Dlx6os1 ( N ). Bars represent mean ± s.e.m. of 9 (I,J,L-N) or 12 (K) replicates from 3 (I,J,L-N) or 4 (K) independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: ChIP-sequencing, Blocking Assay, Binding Assay, Staining, Transgenic Assay, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of C-terminally V5-tagged SP9 constructs: wild-type (WT) SP9, an N-terminal deletion mutant (NΔSP9), and a zinc finger (ZF) domain deletion mutant (SP9ΔZF). The nine-amino-acid transactivation domain (9aaTAD), the three ZF domains, and the position of the patient-derived SP9*378 variant are indicated. B , Co-immunoprecipitation (CoIP) from nuclear lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT, SP9*378-V5, or NΔSP9V5. Left: input fractions probed with anti-SP9 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-SP9 (top) and anti-DLX5 (bottom). C , CoIP from whole-cell lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT or SP9ΔZF-V5. Left: input fractions probed with anti-V5 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-V5 (the anti-SP9 epitope lies within the deleted ZF region, requiring V5 detection; top) and anti-DLX5 (bottom). D , Luciferase reporter activity driven by the hs298 enhancer of Dlx6os1 in N2A cells co-transfected with Dlx5 and either Sp9 , Sp9*378 , N Δ Sp9 , or Sp9 Δ ZF . Bars represent mean ± s.e.m. of9replicatesfrom3independentbatchesperformedintriplicate; pointsindicatebatchmeans. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. E , Label-free quantitative mass spectrometry (MS) of proteins captured by DNA pull-down using a 46-bp fragment of the hs298 enhancer of Dlx6os1 versus the same fragment with the TAATT motifs scrambled, from E14.5 ganglionic eminence (GE) nuclear lysates. Proteins with p ≤ 0.05 and log 2 LFQ FC ≥ 1.5 were considered significantly enriched (n = 25). Transcription factors (TFs) are indicated in black. F , Volcano plot of proteins enriched in anti-SP9 CoIP-MS from E14.5 GE nuclear lysates, relative to IgG controls. Proteins with log 2 FC ≥ 1.5 and p ≤ 0.05 were considered significantly enriched (n = 223). Components of HDAC1/2-containing complexes are highlighted in blue; TFs are indicated in black. 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. G , Gene Ontology (GO) molecular function enrichment analysis of proteins identified by anti-SP9 CoIP-MS. H , Heatmap of ChIP-seq signal intensity across SP9-bound regions for SP9 ( ; ), DLX2, histone modifications (H3K27ac, H3K4me1, H3K4me3) , GTF2I , and NuRD complex subunits (MBD3, CHD4, RBBP4, RBBP7, HDAC1, HDAC2) . Signal is plotted over a ±5 kb window centered on SP9 peak summits and organized by k-means clustering (5 clusters), separating SP9-bound regions by their co-binding patterns with DLX2, NuRD subunits, and active histone marks. All datasets are from mouse E13.5 GE except GTF2I, which is from E13.5 whole brain.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: Construct, Mutagenesis, Derivative Assay, Variant Assay, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Mass Spectrometry, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of the transcription factor (TF) overexpression strategy in N2A cells. Cells were transfected with SP9 alone or co-expressed with WT DLX5 (overexpression experiment 1, OE1) or with the N-terminal deletion mutant NΔDLX5 (OE2), followed by CUT&RUN profiling. B , Domain organization of SP9, DLX5, and NΔDLX5, highlighting the zinc finger (ZF) and homeobox domains. C , Heatmaps of SP9 CUT&RUN signal (anti-V5) centered on SP9 peak regions (±5 kb), clustered into four groups (C1-C4) by k-means. Conditions: SP9 alone, SP9+DLX5 (OE1), SP9+NΔDLX5 (OE2). D , Aggregate signal plots for each cluster (C1-C4) showing normalized SP9 binding profiles at peak centers under the three conditions in (C). E , Top enriched motifs identified by motif analysis of peaks in each cluster, with associated 𝑃 -values. F , Genomic annotation of SP9-bound peaks across clusters. Left: distribution across promoter, exon, intron, intergenic, and other regions. Right: distance to transcription start sites (TSS). G , Genome browser tracks at three representative loci showing SP9 binding under OE1 and OE2 conditions, illustrating loss of SP9 occupancy upon DLX5 coexpression at SP-motif-containing sites and rescue with NΔDLX5, as well as gain of SP9 occupancy at DLX-motif-containing sites with WT DLX5. H , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with combinations of Sp9 , Dlx5 , and N Δ Dlx5 as indicated. I , Volcano plot of co-immunoprecipitation followed by mass spectrometry (CoIP-MS) using anti-V5 in N2A cells co-transfected with Sp9-V5 and Dlx5-FLAG , relative to IgG controls. Proteins with log 2 FC > 1.5 and p < 0.05 were considered significantly enriched (n = 2254). NuRD complex components are highlighted in blue, transcription factors (TFs) in black, and AP-1 family TFs in green. J , Volcano plot comparing the anti-V5 CoIP-MS interactomes between SP9+DLX5 and SP9+NΔDLX5 conditions in N2A cells, using the same cutoffs as in (I). I,J , 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. K , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with varying amounts (ng plasmid DNA) of SP9 (S) and DLX5 (D), showing dose-dependent transcriptional activation. H-K , Bars represent mean ± s.e.m. of 9 replicates from 3 independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. L , Proposed mechanism. When SP9 levels exceed DLX (SP9≫DLX), SP9 binds directly at GC-rich SP motifs. When SP9 and DLX are comparable or DLX is in excess (SP9≈DLX or SP9 < DLX), DLX5 sequesters SP9 to TAATT-containing DLX motifs through the DLX5 N-terminal domain, away from its direct SP-motif binding sites. NΔDLX5, which cannot interact with SP9, fails to redirect SP9 to DLX motifs, restoring SP9 binding at SP motifs.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: Over Expression, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Mass Spectrometry, Plasmid Preparation, Activation Assay

Journal: Neuroreport

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

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

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

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.

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, Staining, Control, Expressing, Binding Assay

Journal: Neuroreport

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

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.

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

Journal: Neuroreport

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

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

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: Transfection, Staining, Binding Assay

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b positively correlates with mATXN3 expression. (A) N2a cells were transfected with full-length mATXN3 constructs carrying either mutant 88 CAG repeats (F-mATXN3) or 22 CAG repeats (F-ATXN3), and then subjected for Western blotting using ATXN3 and Rad23b antibodies. γ-Tubulin was used as an internal control. (B) Quantitative results from (A) show Rad23b is slightly increased in the F-mATXN3 group. N = 9. (C) N2a cells were transfected F-mATXN3 with control (Cont.) or Rad23b plasmids, and then subjected for Western blotting. (D) Quantitative results from (C) show Rad23b significantly increases mATXN3 aggregates. N = 10. (E) N2a cells were transfected F-mATXN3 with control shRNA(shCont.) or shRad23b plasmids, and then subjected for Western blotting. (F) Quantitative results from (E) show shRad23b significantly decreases mATXN3 aggregates. N = 9. (G) F-mATXN3 plasmids were transfected into WT or Rad23b −/− N2a cells, and then subjected for Western blotting. (H) Quantitative results from (G) show Rad23b −/− significantly increases mATXN3 aggregates. N = 9. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Expressing, Transfection, Construct, Mutagenesis, Western Blot, Control, shRNA

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b promotes cell death in the SCA3 cell model. (A–D) N2a cells were transfected F-mATXN3 with control (Cont.) or Rad23b plasmids, and then subjected for propidium iodide (PI) staining(A) and Western blotting (C). Nuclei were indicated by Hoechest 33342(blue). (B) Quantitative results from (A) show Rad23b significantly increases PI positive cells. N = 6. (D) Quantitative results from (C) show Rad23b slightly increases PARP levels (N = 4), but significantly increases cleaved-caspase 3 expression in SCA3 cells (N = 4). (E–H) F-mATXN3 plasmids were transfected into WT or Rad23b −/− N2a cells, and then subjected for PI staining (E) and Western blotting (G). Nuclei were indicated by Hoechest 33342 (blue). (F) Quantitative results from (E) show Rad23b −/− significantly decreases PI positive cells. N = 6. (H) Quantitative results from (G) show Rad23b −/− slightly decreases PARP levels (N = 4), but significantly decreases cleaved-caspase 3 expression in SCA3 cells(N = 4). ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Transfection, Control, Staining, Western Blot, Expressing

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b directly interacts with mATXN3. (A) Reporter constructs are illustrated, including F-mATXN3 fused with green fluorescent protein(GFP) gene and Rad23b fused with Discosoma red fluorescent protein(DsRed) gene. (B) Reporter constructs were transfected into N2a cells as indicated, and then subjected for fluorescent images (top panels). Rad23b is shown in red, and ATXN3 is shown in green. Nuclei were indicated by Hoechest 33342 (blue). Colocalization was shown by arrows, and plot profile analysis (bottom panel) from the white line showed the distribution of different signals. (C) Reporter constructs used for co-immunoprecipitation (IP) are illustrated. (D and E) Cell lysates overexpressing constructs from (C) were subjected for co-IP using Flag (D) or Rad23b (E) antibodies to pulldown protein complex and using Rad23b or ATXN3 antibodies for immunoblotting (IB). IgG-pull down was used as a control.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Construct, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Control

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b affects mATXN3 accumulation through the UPS pathway. (A and B) F-mATXN3 plasmids were transfected into Rad23b −/− cells, treated with MG132, and then subjected for Western blotting (A). (B) Quantitative results from (A) show Rad23b −/− significantly decreases mATXN3 aggregates, and MG132 significantly blocks the effects of Rad23b −/− to significantly increase mATXN3 aggregates. N = 4. (C) N2a cells were transfected F-mATXN3 with control (Cont.) or Rad23b plasmids, and then subjected for Western blotting. (D) Quantitative results from (C) show Rad23b significantly increases endogenous ubiquitin level. N = 6. (E) F-mATXN3 plasmids were transfected into WT or Rad23b −/− N2a cells, and then subjected for Western blotting. (F) Quantitative results from (E) show Rad23b −/− significantly decreases endogenous ubiquitin level. N = 8. (G) N2a cells were transfected F-mATXN3 with control (Cont.) or different dosage of Rad23b plasmids, and then subjected for co-immunoprecipitation (IP). Flag or Rad23b antibodies were used to pulldown protein complex, and ATXN3 or Ubiquitin antibodies were used for immunoblotting (IB), showing Rad23b enhances mATXN3 ubiquitination level in a dose-dependent manner. ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Transfection, Western Blot, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b facilitates the transport of mATXN3 to the proteasome. N2a cells were transfected F-mATXN3 with control (Cont. A) or Rad23b (C) plasmids, and then subjected for immunofluorescent staining. (A) Immunofluorescent images show the signals of Rad23b (red), mATXN3 (green) and PSMB5 (blue, a marker for proteasome localization) after overexpression of F-mATXN3 and control plasmids in N2a cells. The white line was analyzed by the plot profile analysis in (B). (C) Immunofluorescent images show the signals of Rad23b (red), mATXN3 (green) and PSMB5 (blue) after overexpression of F-mATXN3 and Rad23b plasmids in N2a cells. The white line was analyzed by the plot profile analysis in (D). (E) Pearson’s correlation coefficient (PCC) was performed using Pearson’s analysis in the plot profiles, revealing Rad23b increases the PCC value between PSMB5 and mATXN3 signals. N = 3.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Transfection, Control, Staining, Marker, Over Expression

Journal: Neurotherapeutics

Article Title: Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3

doi: 10.1016/j.neurot.2026.e00928

Figure Lengend Snippet: Rad23b disrupts proteasome catalytic functions. (A–D) N2a cells were cotransfected F-mATXN3 and Ub G76V -GFP reporter with Rad23b or Control (Cont.) plasmids, and subjected for fluorescent images and Western blotting. (A) Fluorescent images show the expression of Ub G76V -GFP reporter after different treatments as indicated. Nuclei were indicated by Hoechest 33342 (blue). (B) Quantitative results from (A) show Rad23b significantly increases Ub G76V -GFP signals. N = 6. (C) Western blotting shows the expression of Ub G76V -GFP. γ-tubulin was used as an internal control. (D) Quantitative results from (C) show Rad23b significantly increases Ub G76V -GFP expression. N = 6. (E–H) F-mATXN3 and Ub G76V -GFP reporter were cotransfected into WT or Rad23b −/− N2a cells, and transfected cells were subjected for fluorescent images and Western blotting. (E) Fluorescent images show the expression of Ub G76V -GFP reporter in WT or Rad23b −/− N2a cells transfected with F-mATXN3 and Ub G76V -GFP plasmids. Nuclei were indicated by Hoechest 33342 (blue). (F) Quantitative results from (E) show Rad23b −/− significantly decreases Ub G76V -GFP signals. N = 6. (G) Western blotting shows the expression of Ub G76V -GFP. γ-tubulin was used as an internal control. (H) Quantitative results from (G) show Rad23b Rad23b −/− significantly decreases Ub G76V -GFP expression. N = 5. (I–K) F-mATXN3 was transfected into WT or Rad23b −/− N2a cells, and transfected cells were subjected for determining proteasome activities, including chymotrypsin-like (I), trypsin-like (J) and caspase-like (K) activities, showing Rad23b −/− significantly increases chymotrypsin-like and trypsin-like activities.

Article Snippet: Mouse neuroblastoma N2a cells (ATCC: CCL-131) and Rad23b knockout (Rad23b −/− ) cells were cultured in Eagle’s Minimum Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum (Hyclone) and 1 mM sodium pyruvate (Sigma-Aldrich, P4562).

Techniques: Control, Western Blot, Expressing, Transfection

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