Journal: bioRxiv

Article Title: Differential TDP-43 interactomes between the cortex and cerebellum in the mouse

doi: 10.64898/2026.01.16.699854

Figure Lengend Snippet: TDP-43 differentially interacts with core paraspeckle proteins FUS, PSPC1, SFPQ and NONO in the cortex and cerebellum (a) Representative examples of co-immunoprecipitation of FUS, PSPC1, SFPQ and NONO following TDP-43 pull-down in the cortex and cerebellum of 3-month-old C57Bl/6J mice with both the N (Abcam, #ab225710) and C (Proteintech #12892-1-AP) terminal TDP-43 antibodies. All four proteins were detected in the IP fraction of the cortex with both antibodies, with no band present with IgG alone. In contrast, PSPC1 was absent from the cerebellar samples following pull down with either antibody. IP: immunoprecipitation, FT: flow through. (b) Representative examples of western blot detection of total, nuclear and cytoplasmic FUS, PSPC1, SFPQ and NONO expression levels in the cortex and cerebellum of 3-month-old C57Bl/6J mice. (c-e) Quantification of FUS, PSPC1, SFPQ and NONO total (c), nuclear (d) and cytoplasmic (e) expression levels in the cortex and cerebellum of 3-month-old C57Bl/6J mice. PSPC1 levels were significantly reduced in the nuclear fraction of the cerebellum compared to the cortex. No other significant changes were observed. cx: cortex, cb: cerebellum, n=3 *p<0.05 Welch’s t-test

Article Snippet: Mouse anti FUS (1:500, Santa Cruz: sc-373698), rabbit anti-PSPC1 (1:1000, Abcam: ab133574), mouse anti-SFPQ (1:500, Invitrogen: MA1-25325) and rabbit anti-NONO (1:500, Abcam: ab133574) antibodies were used with loading controls and secondary antibodies as before.

Techniques: Immunoprecipitation, Western Blot, Expressing

Journal: bioRxiv

Article Title: Differential TDP-43 interactomes between the cortex and cerebellum in the mouse

doi: 10.64898/2026.01.16.699854

Figure Lengend Snippet: NONO shows differential cellular distribution between the cortex and cerebellum. (a-d) Representative examples of paraspeckle proteins (PSPr), FUS (a), PSPC1 (b), SFPQ (c) and NONO (d) distribution and colocalisation with TDP-43 in the motor cortex and Crus 1 region of the cerebellum. Scale bar is 20μm. All four paraspeckle proteins show abundant staining in the nucleus, with minimal evidence of expression within the cytoplasm with the exception of NONO within the cerebellum, which shows a distinct punctate pattern of staining within the nucleus and cytoplasm. TDP-43 shows abundant nuclear colocalisation with all four paraspeckle proteins in both the cortex and cerebellum, but no robust evidence of colocalization in the cytoplasm

Article Snippet: Mouse anti FUS (1:500, Santa Cruz: sc-373698), rabbit anti-PSPC1 (1:1000, Abcam: ab133574), mouse anti-SFPQ (1:500, Invitrogen: MA1-25325) and rabbit anti-NONO (1:500, Abcam: ab133574) antibodies were used with loading controls and secondary antibodies as before.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Differential TDP-43 interactomes between the cortex and cerebellum in the mouse

doi: 10.64898/2026.01.16.699854

Figure Lengend Snippet: TDP-43 interactions with FUS, PSPC1, SFPQ and NONO show distinct differences between different cortical and cerebellar cell populations (a) Representative examples of proximity ligation assay (PLA) assessment of TDP-43 with FUS, PSPC1, SFPQ and NONO. (b) Quantitative analysis of PLA spot counts in the large cells of the motor cortex (MC; layer V), and the large (Purkinje cells; PC) and small (Granule cells; GC) of the cerebellum. n=3, *p<0.05; **p<0.01 cytoplasm vs. nucleus; †p<0.05 nucleus vs. motor cortex nucleus #p<0.05; ##p<0.01 cytoplasm vs. motor cortex cytoplasm. Two-way ANOVA with Fisher LSD post hoc. (c) Quantitative analysis of mean PLA spot size in large cells of the motor cortex (MC) and large (PC) and small (GC) cells of the cerebellum. n=3, *p<0.05 vs motor cortex. One way ANOVA with Tukey post hoc

Article Snippet: Mouse anti FUS (1:500, Santa Cruz: sc-373698), rabbit anti-PSPC1 (1:1000, Abcam: ab133574), mouse anti-SFPQ (1:500, Invitrogen: MA1-25325) and rabbit anti-NONO (1:500, Abcam: ab133574) antibodies were used with loading controls and secondary antibodies as before.

Techniques: Proximity Ligation Assay

Journal: Brain

Article Title: Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

doi: 10.1093/brain/awad130

Figure Lengend Snippet: Kinases affect localization and aggregation of non-phosphorylated and phosphorylated Y526 FUS. HEK293T cells were transfected with ( A ) constitutively active c-Src, c-Fyn, and c-Abl expression plasmids and co-transfected with ( B ) constitutively active kinases and a GFP tagged full-length FUS (green). ( C and D ) Quantitative assessment of nuclear and cytoplasmic FUS (magenta) and FUS p-Y526 (red) signal was performed using ImageJ and the ratio of nuclear to cytoplasmic FUS and FUS p-Y526 signals in positively transfected cells was determined alongside their relative cytoplasmic abundance. ( E ) The culture of mouse primary cortical neurons was established, and neurons were transfected with active kinases. Cell nuclei were counterstained with DAPI (blue). One-way ANOVA, * P < 0.05, ** P < 0.01, *** P < 0.001. Scale bars = 20 μm in A and B ; 15 μm in E . ROI = region of interest.

Article Snippet: Consistent with DAB immunohistochemistry, several cortical neurons (10–20%) in FTLD-FUS showed granular cytoplasmic accumulation of FUS p-Y526 , which rarely and only partly co-localized with cytoplasmic FUS aggregates (15–20%) detected with commercial FUS antibody (Novus) ( , white arrow).

Techniques: Transfection, Expressing

Journal: Brain

Article Title: Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

doi: 10.1093/brain/awad130

Figure Lengend Snippet: FUS p-Y526 shows altered nucleocytoplasmic localization in cortical neurons in FTLD-FUS patients. ( A ) DAB immunolabelling using FUS p-Y526 antibody in a representative control section showing diffuse nuclear immunoreactivity, whereas in FTLD-FUS cortical neurons ( right ) showing diffuse nuclear as well as cytoplasmic localization. FUS p-Y526 antibody also recognizes smaller atypical granular (black arrows) and small globular (white arrow) aggregates. ( B ) Co-immunolabelling of FUS p-Y526 and FUS in the cortical neurons of the control and FTLD-FUS patients, ( C ) showing the FUS and FUS p-Y526 signals to only partially overlap in large cytoplasmic inclusions (white arrow). ( D ) Quantification of localization of the fluorescent FUS p-Y526 immunolabelling (Nu = nuclear; Nu & Cyto = nuclear and cytoplasmic; Cyto = cytoplasmic only) by counting neurons in the frontal cortex of control and FTLD-FUS post-mortem brain tissue sections. ImageJ analyses of ( E ) FUS p-Y526 and ( F ) FUS nuclear/cytoplasmic ratio in counted neurons. Statistical analyses were performed using ANOVA. Control n = 4, FTLD-FUS = 4. * P < 0.05, ** P < 0.01. Paraffin sections, scale bar = 18 μm.

Article Snippet: Consistent with DAB immunohistochemistry, several cortical neurons (10–20%) in FTLD-FUS showed granular cytoplasmic accumulation of FUS p-Y526 , which rarely and only partly co-localized with cytoplasmic FUS aggregates (15–20%) detected with commercial FUS antibody (Novus) ( , white arrow).

Techniques: Control

Journal: Brain

Article Title: Abl kinase-mediated FUS Tyr526 phosphorylation alters nucleocytoplasmic FUS localization in FTLD-FUS

doi: 10.1093/brain/awad130

Figure Lengend Snippet: Activity of pAbl increases in the nuclei of FTLD-FUS frontal cortex neurons . ( A ) Co-immunolabelling using FUS p-Y526 and pAbl antibody in the cortical neurons of control and FTLD-FUS patients. ( B ) Counting the frontal cortex neurons with pAbl signal localization in nucleus only (Nu), nucleus and cytoplasm (Nu & Cyto), or in the cytoplasm only (Cyto) in post-mortem tissue sections of control and FTLD-FUS. ImageJ analyses of ( C ) FUS p-Y526 and ( D ) pAbl nuclear/cytoplasmic ratio in counted cortical neurons. Statistical analyses were performed using ANOVA. * P < 0.05, ** P < 0.01. Paraffin sections, scale bar = 20 μm.

Article Snippet: Consistent with DAB immunohistochemistry, several cortical neurons (10–20%) in FTLD-FUS showed granular cytoplasmic accumulation of FUS p-Y526 , which rarely and only partly co-localized with cytoplasmic FUS aggregates (15–20%) detected with commercial FUS antibody (Novus) ( , white arrow).

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: FUS controls muscle differentiation and structure through LLPS mediated recruitment of MEF2 and ETV5

doi: 10.1101/2024.09.18.613669

Figure Lengend Snippet: a-b: Recombinant FUS protein was incubated in presence of ETV5-CFP or/and MEF2A-mCherry recombinant proteins. After immunoprecipitation using FUS antibody then pulled out with A/G-coupled magnetic beads, immunoprecipitated proteins were examined by western blot using FUS, ETV5 and MEF2A antibody. The band intensities of co-immunoprecipitated MEF2A-mCherry and ETV5-CFP were quantified using iBright Analysis Software (B). Error bars represent SEM of four independent experiments. c-d Cooperative binding assay of FUS, ETV5, MEF2A recombinant proteins to FAM labelled Mrpl30 promoter-derived DNA probe. EMSA experiments performed by using increasing concentrations of ETV5 (60, 120, 180 and 240 nM) and constant concentration of FUS (300 nM) and/or MEF2A (50 nM). Panel D shows the percentage of Mrpl30 probe bound across 3 independent experiments. The intensity of the signals of the protein- Mrpl30 probe complex was quantified using the iBright Analysis Software. Error bars represent SEM of three independent experiments. e: Representative confocal images of FUS-EGFP condensates (5 µM) in absence or presence of ETV5-CFP (3 µM) and/or MEF2A-mCherry (0.85 µM) upon protease-mediated cleavage of the MBP-solubility tags. No condensates were observed for ETV5 or MEF2A alone or mixed together in absence of FUS condensates. Bar, 10 µm. f-g: Quantification of FUS-EGFP condensate roundness (F) and size (G). Mean ± SEM of three independent replicates with at least 179 condensates in total from ≥3 different fields of view analyzed. * p<0.05, *** p<0.005 by one-way ANOVA with Tukey’s multiple comparison test. h: Light units relative to Empty vector in C2C12 cells 24 h after transfection of 3xMEF2 luciferase plasmid and an expression plasmid for either hFUS WT, hFUS R495X, LLPS deficient hFUS (hFUS YnCA), LLPS-deficient hFUS with SV40 NLS or empty control plasmid. Nested one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001, **P<0.01, *P < 0.05. Each dot represents the mean of an individual experiment (n=3), each consisting of 4 technical replicates. Data represent the mean ± SEM. Expression of human wild type FUS but neither R495X nor LLPS deficient FUS increases activity of the MEF2 reporter.

Article Snippet: FUS recombinant protein (1 μg; Novus biologicals NBP1-42462) was incubated overnight at 4°C on a rocking platform in presence of ETV5-CFP (1 μg), MEF2A-mCherry (1 μg) and 20 μl of FUS antibody (Santa Cruz sc-47711) or control IgG antibody (Jackson # 115-007-003) in buffer containing 75 mM Hepes pH 7,3, 100 mM KOAc, 1% NP-40, 2,5% glycerol, 0,5 mM DTT and PIC 1X.

Techniques: Recombinant, Incubation, Immunoprecipitation, Magnetic Beads, Western Blot, Software, Binding Assay, Derivative Assay, Concentration Assay, Solubility, Comparison, Plasmid Preparation, Transfection, Luciferase, Expressing, Control, Activity Assay

Journal: Scientific Reports

Article Title: A directional 3D neurite outgrowth model for studying motor axon biology and disease

doi: 10.1038/s41598-021-81335-z

Figure Lengend Snippet: Antibodies and buffers used in this study.

Article Snippet: FUS , Novus Biologicals , NB100-2599 , 1:100 , Buffer 1.

Techniques: