mouse α syn  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Negative stain EM images of α -syn fibrillar polymorphs XG, FM, Ri, F65, and F91 (left) and the C-terminally truncated F110 (right) (scale bar 200 nm). The corresponding α -syn sequence is indicated above. ( B ) Representative immunoblots of supernatant (S) and pellet (P) fractions after a 16 h disaggregation reaction of α -syn polymorphs incubated with the active (+ATP) and inactive (−ATP) chaperone machinery (Hsc70, DnaJB1, Apg2). ( C ) Quantification of released α -syn protein in the supernatant as a fraction of the total (P + S) after 16 h disaggregation by the active chaperone machinery. ( D ) Representative data of a Thioflavin T (ThT)-based disaggregation assay of the polymorphs (XG, blue; F91, red; F65, green; FM, yellow; Ri, gray; F110, purple) by the human Hsc70 chaperone machinery. ( E ) Percentage of disaggregation, calculated from ThT assays, after 16 h disaggregation of the different fibrillar polymorphs by the Hsc70 machinery. Data are mean ± s.e.m. Individual datapoints represent the mean of three technical replicates for independent biological replicates (fibril batches). .

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Staining, Sequencing, Western Blot, Incubation

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) SDS-PAGE of α -syn monomers released to supernatant, separated from fibrillar material by centrifugation, after incubation of fibrils on ice for the indicated times. ( B ) Fraction of total fibrils depolymerized, as shown in ( A ), as a function of incubation time on ice. ( C ) Correlation plot of disaggregation after 16 h in percent (mean ± s.e.m.) and fraction depolymerized with a linear correlation fit (solid line ( r = 0.72)). ( D ) ThT disaggregation assay with 1× (XG, blue; F91, red; F65, green; FM, yellow; Ri, gray; F110, purple) or 2× (black) chaperone concentration. Representative graphs of three technical replicates are shown.

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: SDS Page, Centrifugation, Incubation, Concentration Assay

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Steady-state anisotropy titration of AF-594-labeled DnaJB1 with different concentrations of α -syn fibril polymorphs (XG; blue, FM; orange, Ri; gray, F65; green, F91; red, F110; purple). ( B ) Correlation of experimentally determined DnaJB1 dissociation constants and disaggregation efficiency (as determined in Fig. ) including (dotted line) and excluding (solid line) polymorph Ri. ( C ) Steady-state anisotropy titration experiment of AF-488-labeled Hsc70 in the presence of DnaJB1 and 2 mM ATP from α -syn fibril polymorphs. ( D ) Correlation of experimentally determined Hsc70 dissociation constants and disaggregation efficiency (as determined in Fig. ) including (dotted line) and excluding (solid line) polymorph Ri. Data are mean ± s.e.m. of at least three biological replicates. .

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Titration, Labeling

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Negative stain EM images of polymorphs F65 unlabeled (left) and AF555-labeled (right) (scale bar 200 nm). ( B ) Representative western blot images of unlabeled (left) and AF555-labeled polymorphs (right) incubated with the chaperone machinery in the presence (+ATP) and absence of ATP (−ATP). ( C ) Ratio of released α -syn protein of AF555-labeled and unlabeled fibrils of all polymorphs. Quantification of protein in the supernatant of the total (P + S) after 16 h disaggregation by the active chaperone machinery in ( B ) (analyzed with ImageJ). Data are mean ± s.e.m., n = 3, except polymorph F91 where n = 2. ( D ) Ratio of normalized fluorescence of denatured AF555-labeled fibrils/monomer in GnHCl compared to labeled fibrils/monomer in buffer. Data are mean ± s.e.m., n = 3, except 60% labeled fibrils and monomer where n = 2. ( E , F ) Sucrose density gradient (10–85%) profile of AF555-labeled monomers ( E ) and chaperones (Hsc70, DnaJB1, Apg2) only ( F ). Sucrose gradient of labeled fibrils (XG, FM, Ri, F65, F91, F110) is shown in ( G ). ( H ) Representative sucrose gradient (10–85%) profile of a polymorph XG, F65, F91, and F110 after incubation of AF555-labeled fibrils with the active chaperone machinery (Hsc70, DnaJB1, Apg2, + ATP) for 20 min, 2 h, 16 h, and 16 h incubation with the inactive machinery (−ATP, No Disaggregation).

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Staining, Labeling, Western Blot, Incubation, Fluorescence

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Representative aggregation curves of α -syn monomers seeded with Fibrils (orange); No Disaggregation (black), Disaggregation (green), No seed (gray), and only chaperones (purple) samples for the polymorphs FM (left) and F65 (right). ( B ) Quantification of the initial rate of the seeded aggregation reactions in ( A ), normalized to the initial rate of the fibril only reaction. ( C ) Centrifugation procedure to separate different disaggregation reaction products. A total disaggregation reaction is centrifuged at 3600 × g for 15 min, fibrils (orange) are separated in the pellet and small fragments/oligomers remain in the supernatant (left). By centrifugation of the total reaction at a higher speed (435,630 × g ) for 30 min, small fragments/oligomers (blue) and fibrils are pelleted and only monomers (red) remain in the supernatant (right). ( D ) Representative EM images of a disaggregation and No disaggregation reaction (total reaction; middle), small fragments/oligomer fraction (left) and monomer fraction (right); scale bar 500 nm. ( E ) Representative aggregation curves of α -syn monomers seeded with a total disaggregation reaction (green) and fractionated small fragments/oligomer (blue) and monomer (red) fraction of polymorph FM (left) and F65 (right). ( F ) Quantification of the initial rate of the seeded aggregation reactions in ( E ), normalized to the initial rate of the total disaggregation reaction. ( G ) Representative aggregation curves of α -syn monomers seeded with fractions of a non-disaggregated (−ATP) reaction mixture (total, green; fragments/oligomer, blue; monomer, red). ( H ) Quantification of initial rate of seeded aggregation reactions as shown in ( G ) normalized to the total disaggregation reactions. Data are mean ± s.e.m. Individual datapoints represent the mean of two technical replicates for three independent biological replicates. .

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Centrifugation

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Experimental setup of an in cellulo seeding assay. Three different reaction mixtures are analyzed after 16 h incubation at 30 °C: fibrils only (Fibrils), fibrils incubated with the chaperone machinery (Hsc70, DnaJB1, Apg2) in the absence (No Disaggregation), and presence of ATP (Disaggregation). In addition, the disaggregation sample is further fractionated by differential centrifugation as described in Fig. to generate “Fragments/Oligomers” and “Monomers” fractions. The reaction mixtures are added as seeds to HEK293T cells stably expressing α -syn A53T-YFP, and the number of cells with fluorescent foci is counted. ( B ) Representative fluorescence microscopy images of reporter HEK293T cells seeded with treated fibrils of polymorphs FM and F65. Untreated cells are shown in the lower right (scale bar 20 µm). Fibrils only, fibrils incubated with chaperones in the absence (No Disaggregation) and presence of ATP (Disaggregation), as well as small fragments/oligomer and monomer fractions of the disaggregation reaction separated by centrifugation as described in Fig. . ( C ) Quantification of the percentage of cells with foci normalized to the No disaggregation sample (No Disaggregation, black; Disaggregation, green; small fragments/oligomers, blue; monomers, red; fibrils, orange). Data are mean ± s.e.m. of three biological replicates. Statistical analysis was performed by nonparametric two-way ANOVA with pairwise comparisons of estimated marginal means with Tukey correction for multiple comparisons. Exact p values are indicated in ( C ). .

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Incubation, Centrifugation, Stable Transfection, Expressing, Fluorescence, Microscopy

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Representative fluorescence intensity microscopic images of HEK293T cells stably expressing α -syn A53T-YFP seeded and treated with fibril preparations of polymorph XG, Ri, F91, and F110. The cells were exposed to fibrils only, fibrils incubated with chaperones in the absence (No Disaggregation) and presence of ATP (Disaggregation), as well as small fragments/oligomer and monomer fractions of the disaggregation reaction separated by centrifugation as described in Fig. . Scale bar corresponds to 20 µm. ( B ) Percentage of cells with foci for the individual polymorphs ( n = 3). ( C ) Quantification of the percentage of cells with foci normalized to the No disaggregation sample (No disaggregation, black; Disaggregation, green; small fragments/oligomers, blue; monomers, red; fibrils, orange). The Ri polymorph did not induce aggregation in the used reporter cell system (in ( B )) and was therefore not further analyzed. Data are mean ± s.e.m., n = 3. Statistical analysis was performed by nonparametric two-way ANOVA with pairwise comparisons of estimated marginal means with Tukey correction for multiple comparisons. Exact p-values are indicated in the figure.

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Fluorescence, Stable Transfection, Expressing, Incubation, Centrifugation

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Representative fluorescence intensity microscopic images of HEK293T cells stably expressing α -syn A53T-YFP seeded with fibrils (polymorphs XG and F110) incubated with different chaperone combinations; fibrils without chaperones as a control, fibrils with the whole chaperone machinery (Hsc70, DnaJB1, Apg2), Hsc70 and DnaJB1 and Hsc70, DnaJB1 and Apg2 individually. Scale bar corresponds to 20 µm. ( B ) Quantification of the percentage of cells with foci normalized to fibril-only conditions (XG, top; F110, bottom). Data are mean ± s.e.m., n = 3. Statistical analysis was performed by nonparametric two-way ANOVA with pairwise comparisons of estimated marginal means with Tukey correction for multiple comparisons. Exact p-values are indicated in the figure.

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Fluorescence, Stable Transfection, Expressing, Incubation, Control

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Model of disaggregation efficiency (green) as a function of fibril stability ( y -axis) and chaperone binding ( x -axis). The interplay between chaperone binding to the fibrils and fibril stability has to be in the indicated green area for efficient disaggregation, whereas fibrils located in gray areas (high stability and/or low chaperone binding) are resistant to disaggregation. The distribution of fibril polymorphs analyzed in this study is shown in the plot on the right. ( B ) Cartoon representation of in vitro aggregated α -syn polymorph XG, FM, Ri, F91, F65, and F110. The monomeric α -syn sequence consisting of N-terminal domain, NAC-region, and C-terminal domain with proposed chaperone binding sites indicated (Hsc70, blue; DnaJB1, red) based on Wentink et al (top). Known regions of the monomeric α -syn sequence in the polymorphs that are incorporated into β -sheet structures (small arrow) or are protected from hydrogen–deuterium exchange (shaded area) are indicated. In addition, the respective binding strength of the C-terminal binding antibody 10D2 is indicated (scale from light to dark with increasing affinity). Structural information for F65 can thus far only be derived from similarity in the limited proteolysis pattern to the FM polymorph (Fig. ), suggesting similar regions are buried in the fibril core. In particular, the N-terminal region varies in the degree of structure observed between the various polymorphs, coinciding with the proposed binding sites of Hsc70.

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Binding Assay, In Vitro, Sequencing, Derivative Assay

Journal: The EMBO Journal

Article Title: Structural polymorphism of α -synuclein fibrils alters the pathway of Hsc70-mediated disaggregation

doi: 10.1038/s44318-025-00573-3

Figure Lengend Snippet: ( A ) Cryo-electron microscopy-derived structural models of different fibril conformations generated under the FM (PDB: 6SSX and 6SST) (Guerrero-Ferreira et al, ) and XG (PDB: 8RRR and 8RQM) (Monistrol et al, ) aggregation conditions. ( B ) Hydrogen–deuterium exchange of the XG polymorph followed by NMR spectroscopy. NMR resonance intensities after 3 min of HD exchange in deuterated buffer are plotted relative to their non-exchange intensities as a function of the α -syn sequence. The N- and C-terminal sequences exchange rapidly and are thus mostly disordered, while residues 38–96 make up the slow-exchanging fibril core. Data are mean ± s.d., n = 3.

Article Snippet: α -syn (SNCA) monocolonal mouse IgG , Santa Cruz Biotechnologie , Sc-12767.

Techniques: Cryo-Electron Microscopy, Derivative Assay, Generated, Structural Proteomics, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Protective effects of paederoside in rotenone-induced cellular models of Parkinson’s disease

doi: 10.3389/fcell.2025.1631652

Figure Lengend Snippet: Effect of paederoside on α-Syn nitration triggered by rotenone in an N2A/BV-2 co-culture model (n = 3). (A) Conrol group; (B) Rotenone group; (C) Rotenone+0.1 micromolar (μM Paederoside; (D) Rotenone+1 μM Paederoside; (E) Rotenone+10 μM Paederoside; (F) 20 nM rotenone treatment resulted in a significant increase in nitrated α-Syn in the N2A/BV-2 cell co-culture model; (G) Paederoside significantly reduced the levels of nitrated α-Syn induced by rotenone in an N2A/BV-2 co-culture model. *P < 0.05 as compared with rotenone-treated group and # P < 0.05 as compared with control group.

Article Snippet: The primary mouse monoclonal antibody against human α-Syn (nitrated Tyr125, nitrated Tyr133) was procured from Novus Biologicals.

Techniques: Nitration, Co-Culture Assay, Control