Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: The absence of mα-Syn promotes hα-Syn aggregation in primary neurons. (A) hα-Syn exhibits diffuse distribution in WT neurons and forms inclusions (>1 µm) in cell bodies and neurites in a significant proportion of SNCA−/− neurons. β3-Tubulin staining was performed to reveal neurons, and merged images show orthogonal Z-stack projections (Orth. Proj.). (B) Significantly fewer inclusions (>1 µm) are observed in SNCA−/− neurons with restored mα-Syn expression. (C) Significantly less monomeric hα-Syn is detected in nonionic detergent-soluble (Det. Sol.) fractions of lentivirally infected SNCA−/− neurons than in WT counterparts (n = 4 independent experiments). In contrast, nonionic detergent-insoluble (Det. Insol.) fractions of infected SNCA−/− neurons show more monomeric and HMW hα-Syn species, comparable to the detergent-insoluble fractions of WT neurons treated with hα-Syn PFFs. Actin was used to control for equal protein loading. In B and C, hα-Syn and mα-Syn were differentially revealed using the Syn-211 and D37A6 antibodies, respectively. In A–C, the Mann–Whitney test was applied to obtain P values. In A and B, 25 neurons per condition were quantified (n = 3 independent experiments). (D) Nine different anti–α-Syn antibodies (epitopes in Table S1) detect inclusions in SNCA−/− neurons transfected with hα-Syn. (E) hα-Syn inclusions in transfected SNCA−/− neurons are ThS-positive, phosphorylated at S129, and ubiquitinated. Colocalization was confirmed by assessing regression coefficients (R2). (Scale bars: 10 µm in A and B and 5 µm in D and E.)

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Staining, Expressing, Infection, MANN-WHITNEY, Transfection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Similar total mRNA and protein levels of hα-Syn are expressed in WT and SNCA−/− neurons. Seven days postinfection with viruses encoding hα-Syn (+) or empty vector (−), WT and SNCA−/− primary neurons were lysed, and then RNA (A) or total proteins (B) were extracted. (A) Semiquantitative RT–PCR using specific primers against hα-Syn or GAPDH (housekeeping gene for normalization) followed by agarose gel electrophoresis demonstrates that no significant differences in hα-Syn mRNA levels are detected between WT and SNCA−/− primary neurons. (B) Western blotting of neurons lysed and scraped directly into loading buffer shows that no significant differences in total protein levels of hα-Syn are detected across conditions. hα-Syn and mα-Syn were differentially revealed using the Syn211 and D37A6 antibodies, respectively, and actin was used to control for equal protein loading. The Mann–Whitney test was applied to obtain P values. Three independent experiments were performed.

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: hα-Syn inclusions in SNCA−/− neurons grow and incorporate soluble hα-Syn. (A) Immunofluorescence and 3D rendering of inclusions reveals a significant increase in inclusion volume at 48 and 72 h posttransfection in 25 MAP2+ neurons per condition. (B) Similarly, mEOS2–α-Syn inclusions (n = 50 per condition) show a significant increase in inclusion volume 72 h posttransfection. (C) mEOS2–α-Syn inclusions are immunopositive for total α-Syn (Syn-1), pS129–α-Syn (WAKO), and ubiquitin. (D) mEOS2–Syn inclusions are immobile and grow in volume during ∼1 h of live imaging. (Scale bars: 5 µm in A–C and 2 µm in D.) (E) FRAP and photoconversion experiments allow monitoring rates of Din and Dout of inclusions, respectively. (F and G, Upper) Fluorescence before, during, and after inclusion photobleaching (F) or photoconversion (G) is shown. Higher magnification images of yellow boxes are shown, with crossed circles denoting areas of photobleaching (F) or photoconversion (G). (Lower) Average plots (± SD) of normalized FRAP or photoconversion recovery curves (n = 87 or 63 inclusions, respectively). (Scale bars: 2 µm at lower magnification and 1 µm at higher magnification.) Ch, channel. (H) Values for immobile fractions and diffusion coefficients obtained from photoconversion curves (n = 50 inclusions) are significantly lower than those obtained by FRAP. In A, B, and H, the Mann–Whitney test was applied to obtain P values.

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Immunofluorescence, Imaging, Fluorescence, Diffusion-based Assay, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Endogenous β-Syn naturally acts as an inhibitor of hα-Syn aggregation in primary neurons. (A) Immunocytochemistry validates the loss of α- and β-Syn expression in α-Syn–KO, β-Syn–KO, and triple Syn–KO neurons compared to WT neurons. (B) Significantly more β-Syn–KO neurons and triple-KO neurons (one-way ANOVA and Scheffé post hoc analysis) develop inclusions (>1 µm in diameter) compared to non-Tg neurons (mean ± SD, n = 3 independent experiments, 20 neurons per condition). (C) Inclusions formed in β-Syn–KO and triple Syn–KO neurons are pS129–α-Syn–positive. Colocalization was confirmed by assessing regression coefficients (R2). (Scale bars: 20 µm in A and 5 µm in B and C.)

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Immunocytochemistry, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Generation and characterization of Tg mice expressing hα-Syn or hβ-Syn in the absence of mα-Syn. (A) Schematic representation of the strategy used to generate Tg mice expressing hα-Syn in the absence of mα-Syn. First, Tg mice expressing hα-Syn (hα-Syn+/+; blue) under the PDGFβ promoter (35) were crossed with SNCA−/− mice (red) (13) to generate heterozygotes (F1). These mice then were backcrossed with SNCA−/− mice before being self-crossed to generate Tg mice expressing two copies of hα-Syn in the absence of any mα-Syn expression (hα-Syn+/+SNCA−/−, F3). (B–D) Brains of age-matched hα-Syn+/+ or hα-Syn+/+ SNCA−/− Tg mice (with non-Tg and SNCA−/− mice as controls) were lysed. Then, protein from particulate fractions (B), total RNA (C), or total proteins in unfractionated brains (D) were extracted. (B) Western blotting of particulate fractions using two different antibodies against hα-Syn [Syn-211 (Left) and LB-509 (Right)] shows increased HMW species in detergent-insoluble fractions of hα-Syn+/+ SNCA−/− Tg brains, compared with hα-Syn+/+ mice and non-Tg and SNCA−/− controls. (C) Semiquantitative RT-PCR using specific primers against hα-Syn or GAPDH (housekeeping gene for normalization) followed by agarose gel electrophoresis demonstrates that no significant differences in hα-Syn mRNA levels were detected between hα-Syn+/+ and hα-Syn+/+ SNCA−/− Tg mice brains. (D) Western blotting of unfractionated brains (homogenized and then centrifuged at 5,000 × g for 5 min) of hα-Syn+/+ or hα-Syn+/+ SNCA−/− mice showed that no significant differences in hα-Syn total protein levels were noted between the two conditions. hα-Syn and mα-Syn were differentially revealed using the Syn211 and D37A6 antibodies, respectively, and actin was used to control for equal protein loading. (E) hβ-Syn+/+ and hβ-Syn+/+SNCA−/− mice exhibit similar β-Syn levels in cytosolic fractions. β-Syn was not detected in particulate counterparts. Loss of mα-Syn expression in SNCA−/− mice is verified using the α-Syn–specific antibody SA-3400 and actin to assure equal protein loading. The Mann–Whitney test was applied in C–E to obtain P values, and three independent experiments were performed. (F) Compared with non-Tg mice, the brains of both hβ-Syn+/+ and hβ-Syn+/+SNCA−/− mice show increased terminal distribution of β-Syn in the neocortex, striatum, and hippocampus, that weakens upon proteinase K (PK) treatment. No hβ-Syn inclusions were noted across conditions. (Scale bar: 100 µm.)

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: The absence of mα-Syn promotes hα-Syn aggregation in transgenic mice. (A) Significantly less monomeric hα-Syn is detected in cytosolic fractions from hα-Syn+/+SNCA−/− mice than in hα-Syn+/+ counterparts (n = 3 independent experiments), concomitant with the appearance of HMW hα-Syn species in particulate fractions. Loss of mα-Syn expression in SNCA−/− mice was verified using the mα-Syn–specific antibody D37A6, and actin was used to control for equal protein loading. The asterisk indicates a nonspecific band in cytosolic fractions. (B) Significantly more proteinase K (PK)-resistant hα-Syn inclusions/0.1 mm2 are detected in the neocortex and hippocampus (Hipp.) of hα-Syn+/+SNCA−/− mice than in hα-Syn+/+ mice (n = 3 mice per condition). (Scale bars: 100 µm in overviews and 15 µm in insets.) In A and B the Mann–Whitney test was applied to obtain P values. (C and D) Double-positive hα-Syn inclusions for total α-Syn and pS129 α-Syn (C) or synaptophysin (D) are detected in the cortex, striatum, and hippocampus of hα-Syn+/+SNCA−/− mice. Orthogonal (Orth.) Z-stack projections verify the intracellular nature of inclusions. (Scale bars: 5 µm.)

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Transgenic Assay, Expressing, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: mα-Syn interacts with aggregated hα-Syn species and attenuates seeding and spreading. (A) Following immunoprecipitation (IP) of hα-Syn, mα-Syn monomers (M) were detected only in the sample comprising the mixture of mα-Syn M with hα-Syn PFFs, and not with hα-Syn M. The asterisks indicate the heavy and light chains of the antibody used for IP. (B) A dose-dependent response (interaction) was noted by SPR between immobilized hα-Syn PFFs and injected mα-Syn M, but not between the injected mα-Syn M and immobilized hα-Syn M. (C) Mixtures of α-Syn PFFs and monomers of the same species show significantly higher ThT binding at early time points than seen with α-Syn monomers alone or with PFFs of different species (n = 3 independent experiments). Mean ± SD values are shown. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA and Scheffé post hoc analysis). (D) Significantly more pS129–α-Syn inclusions (arrows) are observed in the cingulate cortex and amygdala of WT mice injected with mα-Syn PFFs (n = 5 mice per condition) than in counterparts injected with hα-Syn PFFs or in SNCA−/− mice injected with hα-Syn/mα-Syn PFFs, which show diffuse staining (arrowheads). The Mann–Whitney test was applied to obtain P values. (Scale bar: 100 µm.)

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: Immunoprecipitation, Injection, Binding Assay, Staining, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: α-Syn PFFs preferentially seed the aggregation of monomeric α-Syn of the same species in vitro and in vivo. (A and B) Recombinant hα-Syn (A) or mα-Syn (B) monomers (M) were incubated either alone (20 µM) or with 10% sonicated hα-Syn PFFs or mα-Syn PFFs. TEM analysis of samples at 0, 2, 8, and 72 h shows that mixtures of α-Syn PFFs and monomers of the same species start forming fibrils at earlier time points than α-Syn monomers alone or mixtures with PFFs of different species. (Scale bar: 200 nm.) (C) mα-Syn PFFs were injected into the striatum of WT (nontransgenic) mice, and α-Syn pathology was assessed in different brain regions 1 mo postinjection. Double-immunolabeling for total α-Syn and pS129–α-Syn or pS129–α-Syn and ubiquitin shows that the somatic inclusions found in the amygdala and cortex of WT mice injected with mα-Syn PFFs are phosphorylated at S129 and are ubiquitinated.

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: In Vitro, In Vivo, Recombinant, Incubation, Sonication, Injection, Immunolabeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Induction of de novo α-synuclein fibrillization in a neuronal model for Parkinson’s disease

doi: 10.1073/pnas.1512876113

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: Image analysis and 3D surface reconstruction were performed using the Zen 2012 SP1 (black edition; Carl Zeiss) and Imaris software, respectively. table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody name/catalog number Epitope Source hα-Syn Syn-211/sc-12767 121-125 Santa Cruz Syn-208 89-110 Virginia Lee * 14H2L1 117-125 Life Technologies LB-509/18-0215 115-122 Life Technologies hα-Syn + mα-Syn Syn-1/610787 80-100 BD Laboratory SA3400 117-131 Enzo Life Sciences Ab5336P 108-120 Millipore mα-Syn D37A6 Cell Signaling pS129 α-Syn WAKO/014-20281 WAKO MJF-R13/ab-168381 Abcam Other synucleins β-Syn/sc-136452 Santa Cruz γ-Syn Homemade EP-1646Y (α- β-Syn) 1-50 Gentex FL-140 (α-, β-, γ-Syn)/sc10717 61-95 Santa Cruz ab-6176 (α-, β,- γ-Syn) 11-26 Abcam Markers Actin/ab-6276 Abcam β3-tubulin Santa Cruz Synaptophysin/D35E4 Cell Signaling Synaptobrevin-2/D601A Cell Signaling Ubiquitin/042691GS Millipore MAP2/188 003 Synaptic Systems Open in a separate window * University of Pennsylvania, Philadelphia.

Techniques: