Journal: Neurobiology of disease

Article Title: Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

doi: 10.1016/j.nbd.2022.105969

Figure Lengend Snippet: Fig. 1. α-Syn PFF surface binding screen and identification of C-term blocking antibodies A) Rat primary cortical neurons (14 DIV) were treated with biotinylated- monomeric alpha-synuclein, biotinylated-oligomeric alpha synuclein, and biotinylated-PFF alpha synuclein for 2 h at concentrations of 1μg/ml, 2μg/ml and 5μg/ml. Cultures were fixed and stained for surface α-Syn species (Green) and Map2 (Red). Images from 1μg/ml treatments are shown. PFF α-Syn species has the most significant binding to the neuronal surface whereas control monomeric alpha synuclein species do not. Oligomeric α-Syn binds to a small subset of neurons. Scale bar, 50 um. B) Quantification of total surface α-Syn species signal normalized to total Map2 signal. Robust surface binding with PFF α-Syn species is observed and signal intensity increases with concentration. We selected 1 μg/ml biotinylated-PFF α-Syn for antibody blocking screen. N = 20 wells, Data represent mean ± SEM, ANOVA ****p < 0.0001. C) Diagram of experimental setup. Anti-α-Syn antibody clones at 30μg/ml and bio-PFF α-Syn at 1μg/ml were added to primary rat cortical neurons and incubated for 2 h and assessed for PFF surface staining. D) Examples images of negative control gp120, and antibody clone hits, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14 DIV) were treated with biotinylated-PFF α-Syn for 2 h and fixed and stained for surface α-Syn species (Green) and Map2 (Red). Antibody clones 31C2 and 56G11 both bind to alpha synuclein at the c-terminal (111−130). Scale bar, 50 um. E) Quantification of total surface α-Syn species signal normalized as percent signal to negative control antibody gp120 for anti-alpha synuclein antibody clones screened. Antibody clones that bind at the C terminal region of α-Syn have strong activity at blocking PFF alpha synuclein binding to neuronal surface. N = 4 wells. Error bar, Data represent mean ± SEM. ANOVA All bar are ****p < 0.0001, except 5 bars indicted at NS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess free unbound alpha synuclein as a measurement of target engagement, a free α-Syn assay was generated on Gyros platform using the drug antibody as capture antibody and rabbit polyclonal anti-alpha synuclein (ProteinTech, 10,842–1-AP) as detection antibody.

Techniques: Binding Assay, Blocking Assay, Staining, Control, Concentration Assay, Clone Assay, Incubation, Negative Control, Activity Assay

Journal: Neurobiology of disease

Article Title: Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

doi: 10.1016/j.nbd.2022.105969

Figure Lengend Snippet: Fig. 2. α-Syn C-terminal antibodies block PFF binding, internalization and induction of pS129 A) Diagram of experimental setup and time course of PFF phenotype development. Anti-α-Syn antibody clones and PFF (1 μg/ml) were added to primary rat cortical neurons and incubated for 10 days and then accessed for PFF surface staining (red), internalization (green), and pS129 induction (white). B) Example images of negative control gp120, and antibody clones, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14 DIV) were treated with biotinylated-PFF α-Syn for 10 days and fixed and stained for surface alpha synuclein PFF (Red), internalized PFF (Green), and Map2 (blue). Antibody clones 31C2 and 56G11 both bind to α-Syn at the c-terminal (111–130), both showed strong blocking of PFF α-Syn surface binding and internalization at 1μg/ml and 10μg/ml of antibody concentration. Scale bar, 50uM. C) Examples images of negative control gp120, and antibody clones, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14DIV) were treated with biotinylated-PFF alpha synuclein for 10 days and fixed and stained for pS129. Antibody clones 31C2 and 56G11 both bind to alpha synuclein at the c-terminal (111–130), and both showed strong blocking of PFF alpha synuclein induced pS129 induction. Scale bar, 50 um. D–F) IC50 curves of antibody clones (gp120, 56G11, 31C2, 52F5) activity in blocking extracellular PFF binding (D), intracellular PFF uptake (E), and PFF pS129 induction (F). N = 4. Error bar, Data represent mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess free unbound alpha synuclein as a measurement of target engagement, a free α-Syn assay was generated on Gyros platform using the drug antibody as capture antibody and rabbit polyclonal anti-alpha synuclein (ProteinTech, 10,842–1-AP) as detection antibody.

Techniques: Blocking Assay, Binding Assay, Clone Assay, Incubation, Staining, Negative Control, Concentration Assay, Activity Assay

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

Article Title: TrkB neurotrophic activities are blocked by α-synuclein, triggering dopaminergic cell death in Parkinson’s disease

doi: 10.1073/pnas.1713969114

Figure Lengend Snippet: α-Synuclein selectively interacts with TrkB receptors. (A) α-Syn specifically interacts with TrkB receptors. GST pull-down assay was conducted from HEK293 cells cotransfected with mammalian GST–α-Syn and HA-Trks. (B) α-Syn N terminus is implicated in binding TrkB. Different mGST-tagged α-Syn truncated were cotransfected with HA-TrkB into HEK293 cells. A GST pull-down assay was performed, and coprecipitated proteins were analyzed by immunoblotting with anti-HA (Top). Schematic diagram of α-Syn truncations (Bottom). (C) TrkB kinase domain is indispensable for α-Syn to interact with TrkB. (Top) Mapping assay for TrkB ICD required for binding to α-Syn. (Bottom) Schematic diagram of TrkB domains. (D) BDNF inhibits α-Syn/TrkB association. Cortical neurons were pretreated with K252a (100 nM) for 15 min, followed by BDNF treatment (50 ng/mL) for 30 min. Coimmunoprecipitation was performed with anti–α-Syn, and the coprecipitated proteins were analyzed by immunoblotting with anti-TrkB (Top). Cell lysates were probed with various antibodies (second through bottom panels). (E) α-Syn associates with TrkB in LBD human patient brains. Brain lysates from LBD patients were immunoprecipitated with control IgG or anti–α-Syn, and the coprecipitated proteins were analyzed by immunoblotting with anti-TrkB. (F) TrkB colocalizes with α-Syn in the LBs of PD patients. Immunofluorescent costainings with anti-TrkB or p-TrkB 706 (Green) and α-Syn (Red) were conducted with human PD brain sections. The nuclei were stained with DAPI. (Scale bar, 20 μm.)

Article Snippet: Primary antibodies to the following targets were used: GST-HRP, alpha-tubulin, beta-actin (Sigma-Aldrich), HA, myc, GFP, PY99, anti–alpha-synuclein, S129, TrkB, TrkC14, Ubiquitin P4D1 (Santa Cruz), anti-Y125, His (GE healthcare), pMAPK/MAPK, pAKT S473/AKT, Fyn, PARP, (Cell Signaling), anti-TH (Sigma-Aldrich), alpha-synuclein LB509 (Thermo Fisher Scientific), and anti-BDNF (Novus Biologicals).

Techniques: Pull Down Assay, Binding Assay, Western Blot, Mapping Assay, Immunoprecipitation, Control, Staining

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: A schematic presentation of the PAC (27M07) used to generate the transgenic mice lines. The organization of the human SNCA locus that is included in the PAC at the top panel. The three distinct SNCA -Rep1 genotypes are indicated below.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Serial analyses to confirm the mouse lines in the study. Presented is an example analysis performed for transgenic line ‘A’, carrying the human SNCA -Rep1 261 bp allele. ( A ) FISH analysis of metaphase chromosomes obtained from the mouse's spleen was used to determine the insertion site and to estimate the range of the copy number. Red-labeled human-PAC 27M07; green-labeled mouse BAC corresponding to mouse chromosome 9, used as a reference for copy number estimation; blue-DAPI. ( B ) RT–PCR to the 27M07 PAC was used to confirm the RNA expression of the human SNCA in the brain of an F1 mouse. ( C ) Western blot using the mouse monoclonal LB509 anti-human-SNCA (Zymed), to confirm the protein expression of the human SNCA in the brain of an F1 mouse. For (B) and (C), each lane represents analysis performed using a mouse-brain sample harvested from the following mouse lines: Rep1 261-A, the 261/261 line ‘A’ transgenic; PAC, control PAC containing the original Rep1 allele transgenic ; Wt, wild type FVB; hSNCA, recombinant human SNCA protein.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Western Blot, Expressing, Control, Recombinant

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Effect of the SNCA -Rep1 promoter genotypes on human SNCA -mRNA expression levels in transgenic mouse brains. ( A ) Validation curve of the Δ real-time assay for relative quantization of human SNCA -mRNA relative to mouse Syp-mRNA in the brain. Relative efficiency plots of human- SNCA and mouse- Syp were formed by plotting the log input amount (ng of total RNA) versus the ΔCt = [Ct( SNCA )−Ct( Syp )]. The slope is 0.045, which indicated the validation of the ΔCt calculation in the range of 0.1–100 ng RNA. ( B ) Fold levels of human SNCA -mRNA were assayed by real-time RT–PCR and calculated relative to mouse Syp -mRNA reference control using the 2 −ΔCt method. No detectable products of the human SNCA -mRNA reaction were observed when WT mouse brain-cDNA was used as template. The risk genotype 261/261 correlates with significant higher SNCA -mRNA levels than the protective genotype 259/259 ( P < 0.0001). The deleted SNCA -Rep1 genotype correlates with significant lower SNCA -mRNA levels than genotypes 261/261 and 259/259 ( P < 0.0001). In total, 72 mice were analyzed; for each genotype (261/261, 259/259 and Δ/Δ), the box plot represents the analysis performed using two transgenic lines, 12 animals from each line (6 males and 6 females), comprising 24 total brain samples per genotype, each of which was analyzed twice independently. The average values are presented by ‘X’. The box plot shows the median (horizontal line inside the box) and the 25th and 75th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile range. The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Expressing, Transgenic Assay, Biomarker Discovery, Quantitative RT-PCR, Control

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Analyzing SNCA proteins in F2 transgenic mouse brains. Representative western blotting ( A ) and ELISA ( B ) results from 24 age- and gender-matched F2 transgenic mouse brains are shown for the three distinct genotypes in six mouse lines. Specimens were assayed with a combination of polyclonal (pAb) and monoclonal (mAb) anti-SNCA antibodies. Equal volumes of whole-brain extracts were either directly loaded onto reducing SDS–PAGE (A) or first adjusted for their total protein content, then diluted at 1: 5000 and subsequently loaded onto ELISA plates (B). Sandwich ELISA (hSA-3/211-B; red bars to the right of each pair) and ELISA (hSA-3/Syn1-B; blue bars on the left of each pair) were carried out on the same 384-well plate in triplicates. Note the near identical monitoring of the recombinant human SNCA protein standards by both assays (left); the absence of any absorbance signal in snca -null (KO) mouse brain by either assay and the absence of signal with the human SNCA-specific ELISA in WT (non-transgenic) mouse brain (center); the fact that the total SNCA signal measuring murine and human protein (blue bars, left) is always stronger than the human-specific signal alone (red bars, right).

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay, Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Sandwich ELISA, Recombinant

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: SNCA - Rep1 expansion increases SNCA protein concentration in mammalian brain. ( A ) Quantification of human SNCA protein in transgenic mouse brain homogenates using sandwich ELISA [hSA-3/211-B] and a 384-well plate format (triplicates; 50 µl/well); a total of 72 mice were analyzed. Concentration values (in ng/µl) were interpolated from the standard curve ( Supplementary Material, Fig. S3A ) and then corrected for the calculated gene copy number in each mouse line. Values were grouped according to the three genotypes examined, i.e. Rep1-261 bp; Rep1-259 bp; Δ-Rep1 (ratio: female:male, 50:50 in all lines). ( B ) Relative ratio of the human SNCA concentration (as shown in A) to the total SNCA protein concentration. The latter was measured using sandwich ELISA [hSA3/Syn1-B] on 384-well plates quantifying both mouse Snca and human SNCA ( Supplementary Material, Fig. S3B ; after correction for gene copy number: Supplementary Material, Fig. S3C ). Bar graph shown in (B) represents values obtained in (A) divided by those shown in Supplementary Material, Fig. S3C .

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Protein Concentration, Transgenic Assay, Sandwich ELISA, Concentration Assay

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Effect of the SNCA -Rep1 promoter genotypes on human SNCA -mRNA expression levels in transgenic mouse whole blood. ( A ) Validation curve of the Δ real-time assay for relative quantization of human SNCA -mRNA relative to mouse Gapdh -mRNA in blood. Relative efficiency plots of human- SNCA and mouse- Gapdh were formed by plotting the log input amount (ng of total RNA) versus the ΔCt = [Ct( SNCA )−Ct( Gapdh )]. The slope is 0.1, which indicated the validation of the ΔCt calculation in the range of 0.1–100 ng RNA. ( B ) Fold levels of human SNCA -mRNA were assayed by real-time RT–PCR and calculated relative to mouse Gapdh -mRNA reference control using the 2 −ΔCt method. No detectable products of the human SNCA -mRNA reaction were observed when WT mouse blood-cDNA was used as template. The risk genotype, 261/261, correlates with higher SNCA -mRNA levels than the protective genotype, 259/259. The deleted SNCA -Rep1 genotype shows no significant correlation with SNCA -mRNA levels in comparison with genotypes 261/261 and 259/259 ( P = 0.524). In total, 72 mice were analyzed; for each genotype (261/261, 259/259 and Δ/Δ), the box plot represents the analysis performed using two transgenic lines, 12 animals from each line (6 males and 6 females); overall, 24 whole-blood samples (per genotype) were each repeated twice, independently. The average values are presented by ‘X’. The box plot shows the median (horizontal line inside the box) and the 25th and 75th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile range. The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Expressing, Transgenic Assay, Biomarker Discovery, Quantitative RT-PCR, Control, Comparison

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: SNCA - Rep1 expansion does not lead to detectable SNCA protein concentration differences in mouse blood. ( A ) Quantification of human SNCA protein in transgenic mouse blood homogenates using sandwich ELISA [hSA-3/211-B] and 384-well plate format (triplicates; 50 µl/well); a total of 72 mice were analyzed. Signals were specific for human SNCA protein because whole blood from WT and snca -knockout mice did not generate ELISA signals above the background ( Supplementary Material, Fig. S4A ). Values were then corrected for the calculated gene copy number in each mouse line and grouped according to the three genotypes examined, i.e. Rep1-261 bp; Rep1-259 bp; Δ-Rep1 (ratio: female:male, 50:50 in all lines). ( B ) Quantification of total SNCA protein concentration levels in transgenic mouse blood was carried out with sandwich ELISA [hSA3/Syn1-B] and 384-well plates. This ELISA measures both mouse snca and human SNCA proteins ( Supplementary Material, Fig. S4B ); values (in ng/µl) were then corrected for gene copy number.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Protein Concentration, Transgenic Assay, Sandwich ELISA, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of characteristic genes in vivo (A) Relative expression levels of DDR-related genes in control and sepsis groups (n=4 in each group). (B) Relative expression levels of ARL4C in control, sepsis, sepsis+Ad-shNC, and sepsis+Ad-shARL4C group (n=8 in each group). (C) Expression levels of inflammatory factors (IL-1β, TNF-α, IL-10, and IL-18) in the peripheral blood of rat with sepsis+Ad-shNC and sepsis+Ad-shARL4C (n=8 in each group). (D) Survival status of rats in each group (n=10 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vivo, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of ARL4C in vitro . (A) Flow cytometry detected the apoptosis rate in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group) (B) Flow cytometry detected the ROS production in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group), *** p < 0.001, **** p < 0.001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vitro, Flow Cytometry, Control

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: The capacity of human or mouse recombinant monomeric aSyn (WT or modified by specific PTMs) to be detected by pS129 commercial antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, and GTX) or the LASH-EGT homemade pS129 antibody was assessed by WB ( a , b , d ) or ELISA ( c ). a , b Forty nanograms of human recombinant monomeric aSyn unphosphorylated (WT) or phosphorylated at the S129 residue (pS129) or di-phosphorylated (pY125/pS129) were detected by WB using total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein) combined with either a commercial (MJF-R13 or pSyn#64) and homemade (LASH-EGT) pS129 antibodies or b commercial pY125 antibody (BD) or the homemade antibody LASH-EGT-pY125. c ELISA confirmed that MJF-R13, but not 81A, is no longer able to detect pS129-aSyn when aSyn was di-phosphorylated (pY125/pS129). d , e One-hundred nanograms of unmodified aSyn (WT) or phosphorylated aSyn at S129 and/or Y125 residues (respectively named pS129, pY125 or pS129/pY125) full length or truncated after residues 133 (1-133/pS129) or 135 (1-135/pS129) were detected by WB using pS129 antibodies ( d ) or total aSyn antibodies (SYN-1 or LASH-EGT 1-20) ( e ). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Recombinant, Modification, Enzyme-linked Immunosorbent Assay, Residue, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Preparation of aSyn fibrils carrying both phosphorylation and nitration modifications. aSyn fibrils were first site-specifically phosphorylated at S129 residue by being incubated with PLK3. Afterwards, TNM was added to induce the nitration of the phosphorylated aSyn fibrils. b The capacity of aSyn fibrils site-specifically phosphorylated at S129 and/or nitrated to be detected by the pS129 commercial antibodies (pSyn#64, MJF-R13, 81A, and EP1536Y) or the LASH-EGT homemade pS129 antibody was assessed by slot blot ( c ) and WB ( d ) analyses. a , b were created using BioRender.com. c , d Thirty-six nanograms of aSyn fibrils (PFFs) that were unmodified (WT), phosphorylated (P), nitrated (N), or nitrated/phosphorylated (N/P) were detected by slot blot ( c ) and WB ( d ) using pS129 antibodies (top panel) in combination with total aSyn antibodies (1–20 and 34–45, bottom panel). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Phospho-proteomics, Nitration, Residue, Incubation, Dot Blot, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: After 14 days of treatment with 70 nM of mouse aSyn PFFs or PBS buffer (negative control), primary hippocampal neurons were fixed or lysed, and ICC ( a – c ) or WB ( d ) analyses were performed using pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, and GTX or LASH-EGT). a – c Newly formed fibrils were detected by confocal imaging ( a ) and quantified by a high-throughput wide-field cell imaging system as previously described . b , c Neurons were counterstained with MAP2 antibody and the nucleus with DAPI staining. Scale bar = 10 μM. b The left-hand side part of the histogram shows the background of each pS129 antibody in PBS-treated primary neurons, while the right-hand side part of the histogram shows the pS129 level detected by the pS129 antibodies in PFFs-treated primary neurons. c pS129 level in PFF-treated primary neurons was re-evaluated after subtracting the pS129 background level from the PBS-treated neurons. The graphs represent the mean +/− SD of three independent experiments. * p < 0.01, *** p < 0.0001 (ANOVA followed by Tukey HSD post hoc test, PBS vs. PFF-treated neurons). d WB analyses of the insoluble fractions of the PBS- and PFF-treated neurons. Membranes were then counterstained by total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein), and actin was used as a loading control. The red arrows indicate the pS129-aSyn positive bands. The blue arrows indicate the undefined bands. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Negative Control, Imaging, High Throughput Screening Assay, Staining, Control, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a , b Sequential biochemical extraction was performed on aSyn KO and WT primary hippocampal neurons cultured for 14 days (DIV14), and the pS129 level was assessed in the nuclear (N), cytosolic (C), and membrane (M) fractions ( a ) or in the soluble (sol) and insoluble (insol) fractions ( b ) by WB using the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Forty nanograms of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls. The pS129 antibodies were used at a dilution of 1/1000. Membranes were then counterstained by total aSyn antibodies (SYN-1 or Fl-140 α/β/γ synuclein). aTubulin, Laminin B, and E-Cadherin were used as loading control for the cytosolic, nuclear, and membrane fractions, respectively ( a ). Actin was used as a loading control for the soluble and insoluble fractions ( b ). All blots were derived from the same experiment and were processed in parallel.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Extraction, Cell Culture, Membrane, Recombinant, Residue, Control, Derivative Assay

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Brain coronal sections (50 μM thick) from different brain areas of aSyn KO mice (Amygdala, Striatum, Motor Cortex, Hippocampus and Substantia Nigra) have been examined to assess the different staining patterns of the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Scale bar = 100 μM. b pS129 level of detection in the soluble and insoluble fractions extracted from the aSyn KO mice brains using the six pS129 antibodies (pSyn#64, MJF-R13, 81a, EP1536Y, GTX, and LASH-EGT). Sequential biochemical extraction was performed on different brain regions (cortical/CTX; Midbrain/MID and Striatum/STR) derived from aSyn KO mice, and the level of pS129 was assessed in the soluble (0.1% Triton-soluble fraction) and the insoluble fraction (2% SDS-soluble fraction). 40 ng of recombinant aSyn WT or phosphorylated at residue S129 (pS129, indicated by the red arrow) were used as positive controls. Membranes were counterstained for total aSyn (SYN-1 or D37A6), and actin was used as a loading control. The red arrows indicate the pS129-aSyn-positive bands. The blue arrows indicate the undefined bands. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Staining, Extraction, Derivative Assay, Recombinant, Residue, Control

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a Brain coronal sections (50 μM thick) from different brain areas of C57BL6/J PFF-injected mice (Amygdala, Striatum, Motor Cortex), 3 months post-injection, have been examined to assess the different staining patterns of the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX, and LASH-EGT). Scale bar = 100 μM. Quantification for each area has been carried out using Qupath and was expressed as a percentage of positive area stained of total ROI. Data are presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 according to one-way ANOVA followed by the Tukey test for multiple comparisons. b Microphotographs of the amygdala were stained with the six pS129 antibodies (pSyn#64, MJF-R13, 81A, EP1536Y, GTX and LASH-EGT) at higher magnification. Scale bar = 10 μM.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: Injection, Staining

Journal: NPJ Parkinson's Disease

Article Title: Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

doi: 10.1038/s41531-022-00388-7

Figure Lengend Snippet: a The C-terminal domain of aSyn remains disordered and accessible in most aSyn species, from monomers to oligomers, fibrils, and Lewy bodies; monomeric forms of aSyn are predominantly disordered but adopt a-helical rich conformations when bound to vesicles. Despite the absence of an atomic model for aSyn oligomers, several types of aSyn oligomers of distinct conformations (disordered or b-sheet rich) and morphological properties have been observed during aSyn in vitro aggregation . Near-atomic models for aSyn fibril core structures solved using cryo-EM (depicted model PDB:6CU7, EMDB: EMD-7618) . On top, a schematic depiction of the fibrils showing the amyloid core sequence (green), disordered N-terminus (gray), and disordered C-terminus (orange). In all cases, the protein’s C-terminus, which harbors various sites for the protein’s truncation and/or phosphorylation, is disordered and accessible. aSyn fibrils found in pathological aggregates are ubiquitinated (black dots), mostly in the N-terminal domain, and phosphorylated, especially at S129 (red dots), and nitrated at multiple tyrosine residues (Y125/Y133/Y136). In LB, modified aSyn fibrils represent one of the key components of Lewy bodies and interact with a large number of proteins, membranous structure organelles, and lipids. Many of these interactions are mediated by N- and C-terminal domains that harbor several sites of PTMs. Recent studies demonstrated that these PTMs play key roles in regulating aSyn fibrils remodeling and LB formation and maturation , . b A selection of aSyn fibrils’ cryo-EM core structures to illustrate the diversity of aSyn fibril structures and the positions of other disease-relevant PTM sites (Y39 in green and S87 in blue) present in the fibril core. One protofilament of the in vitro derived fibrils is displayed, including Polymorph 1a (Pol 1a, PDB-6CU7) , Polymorph 2 (Pol 2, PDB-6SST) and fibrils from pY39 monomers (pY39, PDB- 6L1T) . An example of the fibrils derived from the postmortem brain of multiple system atrophy patients is also displayed (MSA Type I, PDB-6XYO) . The LB representation in a was created using BioRender.com. The fibril core icon “Amyloid fibril” was adapted from BioRender.com.

Article Snippet: The LASH-EGT and the EP1536Y antibodies were excluded from the screen for the following reasons: (1) the LASH-EGT antibody due to its poor capacity to recognize aSyn pathology in PFF-treated neurons (Fig. ); and (2) because the new EP1536Y antibody batches we recently acquired were no longer suitable for ICC, as acknowledged by Abcam by removing the ICC recommendation from their datasheet ( https://www.abcam.com/alpha-synuclein-phospho-s129-antibody-ep1536y-ab51253.html ).

Techniques: In Vitro, Cryo-EM Sample Prep, Sequencing, Phospho-proteomics, Modification, Selection, Derivative Assay

Journal: IBRO Neuroscience Reports

Article Title: Phosphorylated alpha-synuclein distribution in the colonic enteric nervous system of patients with diverticular disease

doi: 10.1016/j.ibneur.2025.02.009

Figure Lengend Snippet: Comparison of Age, Gender Distribution, and P-aSyn Immunoreactivity Across Control, Diverticulosis (Dose), and Diverticulitis (Ditis) Cohorts. P-aSyn IR: phosphorylated alpha-synuclein immunoreactivity.

Article Snippet: Briefly, after deparaffinization in xylol and rehydration in alcohol with descending concentrations, tissue sections were pretreated with citrate buffer (pH 6.0, 95°C, water bath) for 25 minutes, followed by overnight incubation with rabbit-anti-phosphorylated alpha-synuclein antibody (1/1000, D1R1R, Cell signaling Technology) in antibody diluent (Thermo Fisher Scientific, Carlsbad, USA).

Techniques: Comparison, Control