Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) Human SETX transgene expression levels from whole brain RNAs were determined by Taqman qRT-PCR analysis for two month-old SETX-R2136H mice (lines #1917 and #1920). Expression level is shown relative to endogenous murine Setx, and normalized to Gapdh. n = 3 mice/genotype, n = 3 technical replicates.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Expressing, Quantitative RT-PCR

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We immunostained L5 lumbar spinal cord sections from 10 month-old mice of the indicated genotypes for TDP-43, and counterstained with DAPI. Non-Tg and PrP-SETX-wt mice displayed intense TDP-43 staining throughout motor neuron nuclei, but PrP-SETX-R2136H and SETX-L389S+/− mice exhibited reduced TDP-43 nuclear staining and cytosolic mislocalization in motor neurons (boxed). TDP-43 mislocalization was also present in glial cells (arrows). Representative examples of TDP-43 cytosolic aggregation and pronounced nuclear clearing are respectively indicated by white arrowheads and red circle in high power views of motor neurons (lower right). Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Staining

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We prepared L5 lumbar spinal cord sections from 10 month-old mice of the indicated genotypes, performed 3,3′-diaminobenzidine (DAB) staining of TDP-43, and counterstained with hematoxylin to assess TDP-43 mislocalization. Here we see a representative example of a SETX ALS4 mouse motor neuron that has lost TDP-43 nuclear staining. Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Staining

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) HEK293A cells were transfected with a Flag-SETX expression vector, Myc-TDP-43-HA expression vector, and/or a Flag-hnRNP H expression vector as indicated, and then subjected to immunoprecipitation (IP) with an anti-Flag antibody. Immunoblot analysis with the indicated antibodies was performed. Note that anti-TDP-43 immunoblotting of Flag-SETX IPs in lanes 2 and 5 does not detect TDP-43, but immunoblotting of the Flag hnRNP H IP (lane 4) detects both SETX and TDP-43, confirming the validity of the IP technique. No detectable proteins in lanes 1 and 3 rule out artefacts of the Flag IP technique, as these samples did not contain any Flag-tagged proteins.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We immunostained L5 lumbar spinal cord sections from 11.5 month-old mice of the indicated genotypes for Ran, and counterstained with DAPI. The nuclear membrane was usually smooth without invagination in motor neurons from Non-Tg mice, but displayed frequent irregularities in SETX-L389S+/− mice, such as this invagination (yellow arrow). Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques:

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) Primary cortical neurons from Non-Tg, PrP-SETX-R2136H, and SETX-L389S+/− mice were transduced with lentivirus containing a NLS-NES-eGFP shuttling reporter and subjected to photobleaching of the nucleus. Here we see the distribution of eGFP fluorescence in transduced cortical neurons before and after photobleaching of the nucleus.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Transduction, Fluorescence

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) Human SETX transgene expression levels from whole brain RNAs were determined by Taqman qRT-PCR analysis for two month-old SETX-R2136H mice (lines #1917 and #1920). Expression level is shown relative to endogenous murine Setx, and normalized to Gapdh. n = 3 mice/genotype, n = 3 technical replicates.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Expressing, Quantitative RT-PCR

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We immunostained L5 lumbar spinal cord sections from 10 month-old mice of the indicated genotypes for TDP-43, and counterstained with DAPI. Non-Tg and PrP-SETX-wt mice displayed intense TDP-43 staining throughout motor neuron nuclei, but PrP-SETX-R2136H and SETX-L389S+/− mice exhibited reduced TDP-43 nuclear staining and cytosolic mislocalization in motor neurons (boxed). TDP-43 mislocalization was also present in glial cells (arrows). Representative examples of TDP-43 cytosolic aggregation and pronounced nuclear clearing are respectively indicated by white arrowheads and red circle in high power views of motor neurons (lower right). Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Staining

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We prepared L5 lumbar spinal cord sections from 10 month-old mice of the indicated genotypes, performed 3,3′-diaminobenzidine (DAB) staining of TDP-43, and counterstained with hematoxylin to assess TDP-43 mislocalization. Here we see a representative example of a SETX ALS4 mouse motor neuron that has lost TDP-43 nuclear staining. Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Staining

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) HEK293A cells were transfected with a Flag-SETX expression vector, Myc-TDP-43-HA expression vector, and/or a Flag-hnRNP H expression vector as indicated, and then subjected to immunoprecipitation (IP) with an anti-Flag antibody. Immunoblot analysis with the indicated antibodies was performed. Note that anti-TDP-43 immunoblotting of Flag-SETX IPs in lanes 2 and 5 does not detect TDP-43, but immunoblotting of the Flag hnRNP H IP (lane 4) detects both SETX and TDP-43, confirming the validity of the IP technique. No detectable proteins in lanes 1 and 3 rule out artefacts of the Flag IP technique, as these samples did not contain any Flag-tagged proteins.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) We immunostained L5 lumbar spinal cord sections from 11.5 month-old mice of the indicated genotypes for Ran, and counterstained with DAPI. The nuclear membrane was usually smooth without invagination in motor neurons from Non-Tg mice, but displayed frequent irregularities in SETX-L389S+/− mice, such as this invagination (yellow arrow). Scale bar = 20 μm

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques:

Journal: Acta neuropathologica

Article Title: Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients

doi: 10.1007/s00401-018-1852-9

Figure Lengend Snippet: (a) Primary cortical neurons from Non-Tg, PrP-SETX-R2136H, and SETX-L389S+/− mice were transduced with lentivirus containing a NLS-NES-eGFP shuttling reporter and subjected to photobleaching of the nucleus. Here we see the distribution of eGFP fluorescence in transduced cortical neurons before and after photobleaching of the nucleus.

Article Snippet: ABI Assays-on-Demand with TaqMan-based probes for human SETX (Hs00981138_m1), mouse Setx (Mm00616677_m1) and Gapdh (Mm99999915_g1) were show to be highly specific with little to no cross reactivity.

Techniques: Transduction, Fluorescence

Journal: Molecular cell

Article Title: Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters

doi: 10.1016/j.molcel.2017.12.030

Figure Lengend Snippet: (A) Dot-blot probed with S9.6 antibody shows fewer R-loops in ALS4 fibroblasts compared to control. A duplicate blot was probed with anti-dsDNA antibody as loading control. S9.6 intensity was normalized to that of the dsDNA and average was shown. (n= 3 control & 3 patients; error bars are SEM). * represents P<0.05 (t-test). (B) Immunofluorescence staining of ALS4 fibroblasts shows a significant reduction in nuclear S9.6 signal that co-localizes with DAPI (scale bar = 7.5μm) as compared to controls. n = 3 controls & 3 patients, 10 fields of view were counted per sample and quantified using ImageJ. Arrows indicate nuclear R-loops. R-loop staining with the S9.6 antibody shows a significant reduction of R-loop signal in cell nuclei in the (C) lumbar spinal cord and (D) the motor cortex. NeuN is shown as a control of neuronal nuclei staining. Neuronal nuclei indicated by arrows. n = 2 controls & 2 patients; scale bar = 25 m, 10 fields per sample, error bars are SEM. *** represents P<0.001 (t-test). (E) Fewer R-loops in cells transfected with L389S form of SETX compared to WT (t-test, P=0.06). R-loops were analyzed by dot blot using S9.6 antibody. Error bars are SEM of triplicates. S9.6 signal was first normalized to loading control (dsDNA) and then to SETX expression as detected by western blot. (F) Immunofluorescence staining showing reduced nuclear S9.6 signal (green) in cells transfected with L389S form of SETX compared to WT. Note in both L389S and WT SETX transfected cells, S9.6 staining is reduced in those cells with higher SETX expression (red); scale bar = 5 μm, merged image with staining of S9.6, SETX, and DAPI (blue), R-loops in nuclei indicated by arrows.

Article Snippet: TaqMan qPCR assay human SETX , Thermo Fisher Scientific , Cat# Hs00981128_m1.

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

Journal: Molecular cell

Article Title: Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters

doi: 10.1016/j.molcel.2017.12.030

Figure Lengend Snippet: Key Resources Table

Article Snippet: TaqMan qPCR assay human SETX , Thermo Fisher Scientific , Cat# Hs00981128_m1.

Techniques: Control, Recombinant, Transfection, Purification, Reverse Transcription, SYBR Green Assay, Staining, Activity Assay, Methylation, DNA Methylation Assay, Mutagenesis, Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction

Journal: Aging and Disease

Article Title: Collagen XIX Alpha 1 Improves Prognosis in Amyotrophic Lateral Sclerosis

doi: 10.14336/AD.2018.0917

Figure Lengend Snippet: General and clinical characteristics of the study subjects in the blood study cohort.

Article Snippet: Senataxin , SETX , Hs00209294_m1.

Techniques: Biomarker Discovery

Journal: Aging and Disease

Article Title: Collagen XIX Alpha 1 Improves Prognosis in Amyotrophic Lateral Sclerosis

doi: 10.14336/AD.2018.0917

Figure Lengend Snippet: Taqman® probe and primer mixtures used in gene expression assays.

Article Snippet: Senataxin , SETX , Hs00209294_m1.

Techniques: Gene Expression

Journal: Molecular cell

Article Title: Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters

doi: 10.1016/j.molcel.2017.12.030

Figure Lengend Snippet: Key Resources Table

Article Snippet: Halo-SETX plasmid , Promega , Cat# FHC00111.

Techniques: Control, Recombinant, Transfection, Purification, Reverse Transcription, SYBR Green Assay, Staining, Activity Assay, Methylation, DNA Methylation Assay, Mutagenesis, Sequencing, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction