Journal: bioRxiv

Article Title: Altered X-chromosome inactivation predisposes to autoimmune manifestations in mice

doi: 10.1101/2023.04.20.537662

Figure Lengend Snippet: a. Spleen weight of wild-type (WT) and Ftx KO females at 3-months, 1-year and 2-years of age. Median values are shown. ( t-test , * p -values < 0.05). Underneath, representative images of WT and Ftx KO spleens from 3-month, 1-year and 2-year-old females. b. Representative images of hematoxylin-eosin staining on sections of spleens from 1-year-old WT and Ftx KO females. Scale bar; 100 μm. c. Representative flow cytometry analysis of splenic myeloid dendritic cells (m-DC) in WT and Ftx KO 1-year-old females. On the right, percentages of splenic m-DC (CD11b + CD11c + ) and splenic plasmacytoid dendritic cells (p-DC) (CD11c + B220 + SiglecH + ) in leucocytes. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). d. Representative flow cytometry analysis of spontaneously activated B cells (B220 + CD69 + ) (upper panels) or of spontaneously activated T cells (CD4 + CD69 + ) (lower panels) in spleen from 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). e. IgG2a natural antibody levels in sera of 1-year- and 2-year-old WT or Ftx KO females measured by ELISA. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). f. Representative flow cytometry analysis of (B220 + CD138 + ) plasma cells in the spleen of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). g. Representative flow cytometry analysis of (CD19 + B220 lo CD5 + ) natural antibody producing B1a in the peritoneal cavity (PerC) of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). h. Cytokines levels in the blood analysed with CBA assays on sera from 3-month-, 1-year-, or 2-year-old WT and Ftx KO females. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05). i. Representative flow cytometry analysis of monocyte populations including non-classical (CD11b hi CD43 lo Ly6C + ) scavenger monocytes in the spleen of 1-year-old WT and Ftx KO females. Percentages in leucocytes are shown on the graphs beneath. Each triangle represents a mouse. Median values are shown. ( t-test , * p -values < 0.05).

Article Snippet: Bone marrow, spleen, blood and peritoneal cavity cells were stained using the following antibodies: CD3 PerCP-Vio770 (130-119-656, Miltenyi Biotec), CD4-APC (130-123-207, Miltenyi Biotec), CD5-APC-Vio770 (130-120-165, Miltenyi Biotec), CD8-FITC (130-118-468, Miltenyi Biotec), CD11b APC (553312, BD Pharmingen), CD11c PE-Vio770 (130-110-840, Miltenyi Biotec), CD19-FITC (557398, BD Pharmingen), CD21-APC-Vio770 (130-111-733, Miltenyi Biotec), CD23-PE-Vio770 (130-118-764, Miltenyi Biotec), CD38-PE (130-123-571, Miltenyi Biotec), CD43-PE (130-112-887, Miltenyi Biotec), CD69-PE (130-115-575, Miltenyi Biotec), CD138 PE-Vio615 (130-108-989, Miltenyi Biotec), F4/80 FITC (130-117-509, Miltenyi Biotec), Ter119 PE (130-112-909, Miltenyi Biotec), SiglecH APC-Vio770 (130-112-299, Miltenyi Biotec), B220-APC (130-110-847, Miltenyi Biotec), B220 VioBlue (130-110-851, Miltenyi Biotec), IgM-VioBlue (130-116-318, Miltenyi Biotec), IgD-PE (130-111-496, Miltenyi Biotec), GL7-PE-Cy7 (144619, BioLegend), Ly6C-FITC (130-111-915, Miltenyi Biotec) following recommendations of the manufacturers.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Overexpression of α Syn variants in PMN. ( a ) Vector maps of the plasmids used to overexpress EGFP, α Syn-WT, -A30P and -A53T. The respective transcripts are expressed under the control of a human synapsin-1 promoter. ITR: AAV-2 inverted terminal repeat. Int: intron. SV40-pA: SV40 polyadenylation site. WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element. bGH-pA: bovine growth hormone polyadenylation site. ( b and c ) Immunoblots of whole cell protein lysates from PMN transfected with the given plasmids (cells were lysed on DIV 5). In b , an antibody specific for human α Syn (LB509, Invitrogen) was used to detect only the α Syn expressed by the plasmids. In c , total cellular α Syn levels were assessed using an antibody recognizing both human and rat α Syn (BD). At the bottom, quantifications of the band intensities normalized to β -tubulin are shown ( n =3; error bars represent means±S.E.M.; ** P <0.005, *** P <0.0005 according to one-way ANOVA and Dunnett's posthoc test). ( d ) Immunocytochemistry of PMN transfected with the plasmids given on the left side and stained against tyrosine hydroxylase (TH) to identify dopaminergic neurons and human α Syn (LB509, Invitrogen) to check for successful transfection with the respective plasmids (micrographs taken on DIV 5). EGFP is expressed by the plasmid p.EGFP that is either transfected alone or co-transfected with the α Syn-plasmids. Arrows highlight transfected dopaminergic neurons, asterisks mark transfected non-dopaminergic neurons

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Over Expression, Plasmid Preparation, Western Blot, Transfection, Immunocytochemistry, Staining

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Neurite morphology of PMN transfected with different α Syn variants. ( a ) Representative micrographs from an immunocytochemistry of PMN transfected with the plasmids given on the left side and stained against tyrosine hydroxylase (TH); photos were taken on DIV 5. Arrows point at transfected (EGFP positive) dopaminergic (TH-positive) neurons that are also drawn in the neurite scheme on the right side. Asterisks mark transfected non-dopaminergic neurons. ( b–g ) Quantification of single neurite lengths ( b and e ), total neurite length per neuron ( c and f ) and number of primary neurites per neuron ( d and g ) of TH-positive neurons ( b – d ) and TH-negative neurons ( e – g ) transfected with the given plasmids (DIV 5). The data are shown in box plots (box: range from first to third quartile; band inside box: median (=second quartile); star: arithmetic mean value; bottom end of whiskers: minimum of all data; top end of whiskers: 1.5 interquartile range (IQR) of the upper quartile; number given above the upper whisker: number of single values ( n ) included in the respective quantification). ( h ) Quantification of the soma size of PMN transfected with the given plasmids. ( i ) Quantification of the number of TH-positive neurons per view field ( × 10) on DIV 5 after transfection with the given plasmids. Total neuron numbers did not differ significantly among the groups. Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P <0.05, ** P <0.005, *** P <0.0005. Error bars represent means±S.E.M

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Transfection, Immunocytochemistry, Staining, Whisker Assay

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Sholl analysis of PMN transfected with different α Syn variants. ( a ) Representative micrograph of an EGFP-transfected TH-positive neuron with highlighted neurites (purple) and superimposed circles at given distances from the center of the soma. For Sholl analysis, the total number of neurite crossings was counted at each circle with the radius increasing in steps of 12.5 μ m. ( b and c ) Results of the Sholl analysis of TH-positive-neurons ( b ) and TH-negative neurons ( c ) transfected with the given plasmids (DIV 5). The mean number of neurite crossings at a given distance from the center of the soma is plotted. ( d–g ) Additional quantifications of the Sholl analysis of TH-positive neurons ( d and e ) and TH-negative neurons ( f and g ) transfected with the given plasmids (DIV 5). The critical value ( d and f ) is the radius r at which there is a maximum number of neurite crossings ('neurite maximum'). The Schoenen ramification index ( e and g ) is calculated by dividing the neurite maximum (i.e. the maximum number of branches as specified by the critical value) by the number of primary neurites originating from the soma. It represents the degree of ramification of a neurite tree. Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P <0.05, ** P <0.005, *** P <0.0005. Error bars represent means±S.E.M. n =135 randomly chosen neurons per condition from three independent experiments

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Transfection

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Macroautophagy in PMN transfected with different α Syn variants. ( a ) Representative LC3 immunoblots of whole cell protein lysates from PMN transfected with the plasmids given above. As indicated above the blot, the groups on the right side were treated with bafilomycin (1 nM for 6 h) for evaluation of autophagic flux. The photo represents optimal exposition times for detection of the LC3-II band; for quantification of the higher intensity LC3-I band, photographs with lower exposition times were chosen. ( b–d ) Quantifications of the band intensities normalized to actin as loading control are shown for LC3-I ( b ) and LC3-II ( c ). Changes in autophagic flux are displayed in d , where the quotients of the LC3-II/LC3-I quotients after and before bafilomycin treatment are shown for each given plasmid. ( e and f ) Representative p62-immunoblot ( e ) of whole cell protein lysates from PMN transfected with the plasmids given above. Quantifications of the band intensities normalized to actin as loading control are shown in f . ( g ) Representative pseudo-confocal micrographs from an immunocytochemistry of PMN transfected with the plasmids given on the left side, with (+) or without (−) bafilomycin treatment as indicated on the left side and stained against tyrosine hydroxylase (TH) and LC3; photos were taken on DIV 5. ( h and i ) Quantification of the LC3 immunocytochemistry (representative micrographs: see g ). Intraneuronal LC3 puncta were counted in TH-positive neurons ( h ) and TH-negative neurons ( i ) transfected with the given plasmids with (+) or without (−) bafilomycin treatment on pseudo-confocal micrographs (DIV 5). Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P <0.05, ** P <0.005, *** P <0.0005. Error bars represent means±S.E.M. n =3 independent experiments

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Transfection, Western Blot, Plasmid Preparation, Immunocytochemistry, Staining

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Colocalization of LC3 and α Syn in PMN transfected with different α Syn variants. ( a ) Representative confocal micrographs of PMN transfected with the respective α Syn variant given on the left side and immunostained against TH, LC3 and α Syn as specified on top. ( b ) Quantification of colocalization between α Syn and LC3 in the soma of TH-positive neurons after transfection with different α Syn variants represented by means of Li's intensity correlation coefficient. Cells transfected with α Syn-A30P show significantly less colocalization than those transfected with α Syn-WT or -A53T. Statistics: one-way ANOVA followed by Tukey-Kramer post hoc test, *** P <0.0005 as compared with both other groups. Error bars represent means±S.E.M. n =2 independent experiments

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Transfection, Variant Assay

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: Axonal transport of synaptophysin-positive vesicles in PMN transfected with different α Syn variants. ( a ) Representative micrograph showing PMN transduced with AAV.synaptophysin-EGFP (expressing EGFP-tagged synaptophysin) and co-transfected with p. α Syn-WT and p.dsRed (to identify transfected neurons) (DIV 5). In the higher magnification pictures, the EGFP-positive vesicles (arrows) can be seen along the transfected axon. ( b ) Representative kymographs of the movements of EGFP-tagged synaptophysin along neurites transduced with the plasmids given on the left side within 10 s ( y axis). Arrows point at representative moving vesicles, asterisks mark stationary vesicles. ( c–f ) Quantifications of synaptophysin-EGFP transport in neurites transfected with the given plasmids. Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P <0.05, ** P <0.005, *** P <0.0005. Error bars represent means±S.E.M. n =3 independent experiments

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: Transfection, Transduction, Expressing

Journal: Cell Death & Disease

Article Title: Alpha-Synuclein affects neurite morphology, autophagy, vesicle transport and axonal degeneration in CNS neurons

doi: 10.1038/cddis.2015.169

Figure Lengend Snippet: In vivo live imaging of acute axonal degeneration in the rat optic nerve. ( a ) Schematic drawing of the experimental setup. The optic nerve was crushed with a suture and then imaged in vivo on the proximal and distal side of the crush for 6 h. Single axons from retinal ganglion cells were labeled with EGFP expressed by AAV that were injected intravitreally 4 weeks before the imaging. ( b ) Low magnification micrograph of an exposed optic nerve before crush lesion. The suture is loosely tied around the nerve. Single EGFP-positive axons can be identified. ( c and d ) Quantification of the AIR at the given time-points after crush lesion on the proximal ( c ) and distal side ( d ) of the crush ( n =15 axons from three independent experiments per group). Statistics: one-way ANOVA followed by Dunnett's post hoc test, * P <0.05, ** P <0.005, *** P <0.0005. Error bars represent means±S.E.M. ( e ) Representative composite images of projected z-stack micrographs of optic nerves transduced with AAV.EGFP, AAV. α Syn-WT or AAV. α Syn-A30P at the time points after crush lesion given on top

Article Snippet: Membranes were incubated at 4 °C overnight in the following primary antibodies: anti-human- α Syn monoclonal antibody (mAb) (Invitrogen; Cat. No. 328100), 1 : 500; mouse anti- α Syn mAb clone 42 (BD Transduction Laboratories; Cat. No. 610786), 1 : 500; mouse anti-GAPDH mAb clone 6C5 (Biotrend, Köln, Germany), 1 : 2500.

Techniques: In Vivo, Imaging, Labeling, Injection, Transduction

Journal:

Article Title: Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous ?-Synuclein Inclusions of ?-Synucleinopathies

doi: 10.2353/ajpath.2006.050770

Figure Lengend Snippet: List of Antibodies Used in This Study

Article Snippet: 50 Ultrathin sections (70 nm) were cut and double immunolabeled using mouse anti-α-syn monoclonal antibody (mAb) syn303 and rat anti-Hsp90 mAb 9D2 followed by anti-mouse IgG and anti-rat IgG conjugated with 10-nm protein A gold and 18-nm protein L gold (Rockland Immunochemical, Gilbertsville, PA), respectively.

Techniques: Ubiquitin Proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases

doi: 10.1167/iovs.61.6.12

Figure Lengend Snippet: Primary Antibodies

Article Snippet: Anti-pan synuclein (pan α-Syn, mouse) , 1:1000 , BD Biosciences (San Jose, CA, USA) , 610787.

Techniques:

Journal: Investigative Ophthalmology & Visual Science

Article Title: Assessment of the Retina of Plp-α-Syn Mice as a Model for Studying Synuclein-Dependent Diseases

doi: 10.1167/iovs.61.6.12

Figure Lengend Snippet: α-Syn immunoreactivity in retinas of different ages. Human α-Syn immunoreactivity ( green ) and 4′,6-diamidino-2-phenylindole (DAPI) ( blue ) of wild-type (WT) and Plp-α-Syn retinal sections. Distinct α-Syn immunoreactivity was obvious in the IPL and GCL in both age groups of Plp-α-Syn but was absent in WT mice. ( A ) Central and peripheral retina of adult (8 weeks) and aged (12 months) Plp-α-Syn animals. The antibody exclusively detected human α-Syn (anti-human-α-synuclein; see ). The staining intensity increased in aged animals ( arrows ). ( B ) Central retina of adult and aged WT mice. Scale bar : 50 µm.

Article Snippet: Anti-pan synuclein (pan α-Syn, mouse) , 1:1000 , BD Biosciences (San Jose, CA, USA) , 610787.

Techniques: Staining