Journal: Cell Reports

Article Title: USP15 Deubiquitinase Safeguards Hematopoiesis and Genome Integrity in Hematopoietic Stem Cells and Leukemia Cells

doi: 10.1016/j.celrep.2020.108533

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti-FUS antibody , Novus Biologicals , Cat# NB100-565.

Techniques: Recombinant, Gene Knockout, Transfection, Cell Isolation, RNA Sequencing, Knockdown, Mass Spectrometry, Expressing, Knock-Out, Illumina Sequencing, Multiplexing, CRISPR, Control, shRNA, Sequencing, Quantitative RT-PCR, Plasmid Preparation, Software, Quantitative Proteomics, Irradiation, Imaging

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:

Article Title: Extensive FUS-immunoreactive Pathology in Juvenile Amyotrophic Lateral Sclerosis with Basophilic Inclusions

doi: 10.1111/j.1750-3639.2010.00413.x

Figure Lengend Snippet: Histopathology of juvenile ALS with basophilic inclusions in comparison with that of late-onset sporadic ALS. (A, E) H&E sections of the spinal cord show the presence of basophilic inclusions in the remaining motor neurons in Cases 1 and 2. (B, F) Immunohistochemical staining shows that the basophilic inclusions are positive for FUS proteins. (C, G) Furthermore, these inclusions are only weakly positive for ubiquitin, but negative for TDP-43 (D, H). (I) In contrast, spinal motor neurons in patients with late-onset sporadic ALS show no evidence of basophilic inclusions on H&E-stained section. (J) Furthermore, immunohistochemistry shows the presence of FUS protein in the nucleus of remaining spinal motor neurons. (K–L) Many of the spinal motor neurons in late-onset sporadic ALS patients show abnormal accumulation of ubiquitinated proteins (K) and TDP-43 proteins (L). Scale bar in L is 25 µm and applies to all panels.

Article Snippet: The tissues were then incubated with a primary antibody for FUS (1:20, Novus Biologicals) or ubiquitin (1:10, Abcam), and the goat anti-rabbit secondary antibody conjugated with 15-nm gold particles (1:20, Ted Pella, Redding, CA, USA).

Techniques: Histopathology, Comparison, Immunohistochemical staining, Staining, Ubiquitin Proteomics, Immunohistochemistry

Journal:

Article Title: Extensive FUS-immunoreactive Pathology in Juvenile Amyotrophic Lateral Sclerosis with Basophilic Inclusions

doi: 10.1111/j.1750-3639.2010.00413.x

Figure Lengend Snippet: Ultrastructural analyses of basophilic inclusions in juvenile ALS. (A) A low magnification electron micrograph of the intracytoplasmic inclusion in a spinal motor neuron. The three rectangles in panel A highlight areas of higher magnifications shown in panels B, C and D. (B–D) Higher magnification image at the edge and the center of the inclusion. Both panels B and D show aggregates of disorganized endoplasmic reticulum (ER) and mitochondria (M). (C) In contrast, the center of the inclusion contains filamentous structures that measure 15–20 nm in diameter. (E,F) Immunogold EM using FUS-specific antibody shows that the majority of FUS proteins are associated with the filamentous structures (E), whereas the immunogold staining for ubiquitin show very few positive gold particles.

Article Snippet: The tissues were then incubated with a primary antibody for FUS (1:20, Novus Biologicals) or ubiquitin (1:10, Abcam), and the goat anti-rabbit secondary antibody conjugated with 15-nm gold particles (1:20, Ted Pella, Redding, CA, USA).

Techniques: Staining, Ubiquitin Proteomics

Journal:

Article Title: Extensive FUS-immunoreactive Pathology in Juvenile Amyotrophic Lateral Sclerosis with Basophilic Inclusions

doi: 10.1111/j.1750-3639.2010.00413.x

Figure Lengend Snippet: Basophilic inclusions can be detected in cerebral cortex and brainstem nuclei in patients with juvenile ALS. (A–B) An H&E section shows the presence of basophilic inclusion in the cytoplasm of a neuron in the cingulate gyrus (A). IHC using FUS antibody confirms that these inclusions are positive for FUS protein (B). (C–E) The basophilic and FUS-positive inclusions can also be identified in neurons in the reticular formation in the medulla oblongata (C and D) and the red nucleus (E). (F) In contrast, neurons in the hypoglossal nucleus in both juvenile ALS patients show staining of FUS proteins in the nuclei.

Article Snippet: The tissues were then incubated with a primary antibody for FUS (1:20, Novus Biologicals) or ubiquitin (1:10, Abcam), and the goat anti-rabbit secondary antibody conjugated with 15-nm gold particles (1:20, Ted Pella, Redding, CA, USA).

Techniques: Staining

Journal:

Article Title: Extensive FUS-immunoreactive Pathology in Juvenile Amyotrophic Lateral Sclerosis with Basophilic Inclusions

doi: 10.1111/j.1750-3639.2010.00413.x

Figure Lengend Snippet: Comparisons of age onset and neuropathological features of FUS-immunostaining in familial ALS cases with FUS/TLS mutations.

Article Snippet: The tissues were then incubated with a primary antibody for FUS (1:20, Novus Biologicals) or ubiquitin (1:10, Abcam), and the goat anti-rabbit secondary antibody conjugated with 15-nm gold particles (1:20, Ted Pella, Redding, CA, USA).

Techniques: Immunohistochemistry, Ubiquitin Proteomics