Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: (A) Diversity of aSyn pathology in synucleinopathies with (Ai) granular/ punctate cytoplasmic inclusions in the neurons; (Aii) classical LBs in the neuronal soma; (Aiii) LNs in the neuronal processes; (Aiv) astrocytic aSyn accumulations; (Av) oligodendroglial cytoplasmic inclusions. These pathological structures show differences in their positivity to aggregation markers, including ubiquitin (Ub) and p62. Schematic created with BioRender.com (agreement no: QW23G6FJ76 ). (B) Cryo-EM three-dimensional reconstructions of the recombinant full-length aSyn PFFs to show the polymorphism of aSyn fibrils generated in vitro ( ; ). Four distinct polymorphs were identified based on the protofilament fold and inter-protofilament interfaces: Polymorph 1a ‘rod’ (PDB-6CU7, EMD-7618); polymorph 1b ‘twister’ (PDB-6CU8, EMD-7619); polymorph 2a (PDB-6SSX, EMD-10307); and polymorph 2b (PDB-6SST, EMD-10305). (C) aSyn PTMs identified in synucleinopathy brain tissues, which include acetylation, ubiquitination, phosphorylation, nitration and truncation across the whole sequence of the protein. (D) A schematic representation of the steps followed for the generation, characterisation, validation and application of the novel aSyn monoclonal mouse antibodies. These involved ( Di ) antibody design via the selection of immunogens comprising of aSyn recombinant proteins and peptides; ( Dii ) immunisation of the mice followed by lymphocyte-myeloma fusion; ( Diii ) screening of the hybridomas via ELISA, DB and WB, isotyping and subcloning, and ( Div ) acquisition of purified antibodies. These antibodies were then ( Dv ) characterised using a library of aSyn and bSyn recombinant proteins for their epitopes, conformational selectivity, sensitivity, specificity and reactivity via DB and WB. The antibody specificity was then further validated on ( Dvi ) aSyn KO mouse primary hippocampal and cortical neurons, and in aSyn KO mouse tissue of amygdala. ( Dvii ) The antibodies were validated on human brain tissues of different LB disorders. ( Dviii ) The mouse aSyn-reactive antibodies were applied to neuronal seeding model and PFF-injected mouse brain tissues to profile the newly formed aggregates. Schematic created with BioRender.com (agreement no: FN23G6E1SR ). aSyn = alpha-synuclein; bSyn = beta-synuclein; DB = dot blot; cryo-EM = cryogenic electron microscopy; ELISA = enzyme-linked immunoassay; KO = knockout; LB = Lewy body; LN = Lewy neurite; PFFs = pre-formed fibrils; PTM = post-translational modification; Ub = ubiquitin; WB = Western blot

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Ubiquitin Proteomics, Cryo-EM Sample Prep, Recombinant, Generated, In Vitro, Phospho-proteomics, Nitration, Sequencing, Biomarker Discovery, Selection, Enzyme-linked Immunosorbent Assay, Subcloning, Purification, Injection, Dot Blot, Electron Microscopy, Knock-Out, Modification, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Validation and epitope mapping of aSyn antibodies. (A) DB validation of the novel monoclonal, in-house polyclonal and commercially available aSyn antibodies against the N-terminal, non-amyloid component (NAC) and the C-terminal regions of aSyn for epitope mapping, specificity and species reactivity using a selected library of aSyn and bSyn recombinant proteins under native conditions. Protein loading control was run via Ponceau S staining. All loaded proteins represent human aSyn forms except for human bSyn and mouse aSyn full-length (m FL) proteins. Red arrows highlight the sensitivities of the antibodies to the presence of neighbouring aSyn PTMs. (B) A schematic to represent the novel monoclonal (marked with *), in-house polyclonal (marked with **) and pre-existing commercial aSyn antibodies (marked with ***) included in this study. The commercial antibodies developed jointly with Biolegend are marked with *°*. The epitope information of each antibody is indicated in blue. Schematic created with BioRender.com (agreement no: JR23G6G5LA ). (C) Validation of the aSyn PTM antibodies via DB screening. aSyn = alpha-synuclein; bSyn = beta-synuclein; DB = dot blot; FL = full-length; m = mouse; PTM = post-translational modification

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Recombinant, Control, Staining, Dot Blot, Modification

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Validation and epitope mapping of aSyn antibodies. WB validation of the novel monoclonal, in-house polyclonal and commercially available aSyn antibodies against the N-terminal, non-amyloid component (NAC) and the C-terminal regions of aSyn for epitope mapping, specificity and species reactivity using a selected library of aSyn recombinant proteins under denatured conditions. Protein loading control was run via re-blotting the membranes with complementary aSyn antibodies. All loaded proteins represent human alpha-synuclein (aSyn) forms except for human beta-synuclein (bSyn) and mouse aSyn full-length (m FL) proteins. Red arrows highlight the sensitivities of the antibodies to the presence of neighbouring aSyn PTMs. aSyn = alpha-synuclein; bSyn = beta-synuclein; FL = full-length; m = mouse; PTM = post-translational modification; WB = Western blot

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Recombinant, Control, Modification, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Validation of the aSyn PTM antibodies via DB, WB and SPR using human recombinant aSyn standards. In-house and commercial aSyn PTM antibodies were validated by (A) WB screening. Further (B) DB and (C) WB analyses on 6A3-E9 showed that this antibody is specific to human aSyn truncated at residue 120. (D) SPR sensograms showed the binding responses of immobilised antibody 6A3-E9 against varying concentrations of aSyn human 1-120 (top) or aSyn human full-length (bottom). aSyn = alpha-synuclein; DB = dot blot; PTM = post-translational modification; SPR = surface plasmon resonance; WB = Western blot; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Recombinant, Residue, Binding Assay, Dot Blot, Modification, SPR Assay, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Amino acid sequences of aSyn, of its mouse orthologue and its protein homologues. (A) The amino acid sequence of aSyn human and its mouse orthologue. The residue differences between the two proteins are highlighted in red. (B) The synuclein family comprises three homologous proteins – alpha (aSyn), beta (bSyn) and gamma synuclein (gSyn). Only alpha-and beta-synuclein were included in this study. The sequence differences are highlighted in red.

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Selectivity of aSyn antibodies over aSyn conformations. (A) Representative EM images of aSyn human WT monomers, oligomers and fibrils. (B) DB and WB characterisation of the novel monoclonal, in-house polyclonal and commercially available aSyn antibodies to determine their conformational selectivity using aSyn human WT recombinant monomers, oligomers and pre-formed fibrils. aSyn = alpha-synuclein; DB = dot blot; EM = electron microscopy; f = fibrils; m = monomers; o = oligomers; WB = Western blot; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Recombinant, Dot Blot, Electron Microscopy, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of non-modified aSyn antibodies on aSyn KO hippocampal and cortical neurons by ICC. PBS- and PFF-treated aSyn KO (A) hippocampal and (B) cortical neurons were immunostained to validate the specificity of the antibodies with epitopes against the N-terminus, NAC region and C-terminus of aSyn. Blue arrows indicate bSyn positivity in PBS-treated neurons, green arrows indicate positivity to aSyn mouse WT fibrils in PFF-treated neurons, and red arrows indicate non-specific background both in PBS- and PFF-treated neurons. aSyn = alpha-synuclein; bSyn = beta-synuclein; ICC= immunocytochemistry; KO = knockout; MAP2 = microtubule associated protein 2; PBS = phosphate buffered saline; PFF = pre-formed fibril; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Modification, Immunocytochemistry, Knock-Out, Saline

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of aSyn PTM antibodies on aSyn KO hippocampal and cortical neurons by ICC. PBS- and PFF-treated aSyn KO (A) hippocampal and (B) cortical neurons were immunostained to validate the specificity of the antibodies with epitopes against the PTMs of aSyn. Green arrows indicate positivity to aSyn mouse WT fibrils in PFF-treated neurons, and red arrows indicate non-specific background both in PBS- and PFF-treated neurons. aSyn = alpha-synuclein; bSyn = beta-synuclein; ICC = immunocytochemistry; KO = knockout; MAP2 = microtubule associated protein 2; PBS = phosphate buffered saline; PFF = pre-formed fibril; PTM = post-translational modification; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Immunocytochemistry, Knock-Out, Saline, Modification

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of aSyn antibodies on aSyn KO hippocampal and cortical neurons by WB. PBS- and PFF-treated aSyn KO hippocampal and cortical neurons were separated to soluble and insoluble fractions by sequential extraction, and stained using (A) non-modified and (B) aSyn PTM antibodies for specificity validation. Green arrows indicate bands specific to aSyn mouse WT fibrils, blue arrows indicate bSyn-specific bands, and red arrows indicate non-specific background. aSyn = alpha-synuclein; bSyn = beta-synuclein; KO = knockout; PBS = phosphate buffered saline; PFF = pre-formed fibril; PTM = post-translational modification; WB = Western blot; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Extraction, Staining, Modification, Knock-Out, Saline, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of aSyn antibodies on aSyn KO mouse brain tissue by IF. The in-house and commercial antibodies against (A) non-modified aSyn and (B) aSyn PTMs were screened for their specificity using aSyn KO mouse amygdala sections. Red arrows indicate non-specific background. aSyn = alpha-synuclein; IF = immunofluorescence; KO = knockout; PTM = post-translational modification

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Modification, Immunofluorescence, Knock-Out

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Application and validation of the aSyn antibodies on PD tissue. (A) The in-house monoclonal, polyclonal and commercial aSyn antibodies were optimised for IHC on the PD cingulate cortex. (B) Triple labelling of PD cingulate cortex by IF using an aSyn N-terminal (LASH-BL 34-45), a C-terminal (AB 134-138) and a pS129 (BL 81A-biotin) antibody. aSyn = alpha-synuclein; IF = immunofluorescence; IHC = immunohistochemistry; PD = Parkinson’s disease

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Immunofluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: IF labelling of PD cingulate cortex using the aSyn N-terminal LASH-EGTNter 1-20 or LASH-BL 34-45, aSyn C-terminal BL 4B12 103-108 or AB 134-138, and aSyn pS129 BL 81A-biotin antibodies. aSyn = alpha-synuclein; IF = immunofluorescence; IHC = immunohistochemistry; PD = Parkinson’s disease

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Immunofluorescence, Immunohistochemistry

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of the aSyn antibodies against C-terminal tyrosine phosphorylations on aSyn KO hippocampal and cortical neurons by ICC and WB. PBS- and PFF-treated aSyn KO (A) hippocampal and (B) cortical neurons were immunostained to validate the specificity of the Abcam antibodies with epitopes against aSyn pY125, pY133 and pY136. (C) PBS- and PFF-treated aSyn KO hippocampal and cortical neurons were separated to soluble and insoluble fractions by sequential extraction, and stained using Abcam pY125, pY133 and pY136 antibodies for specificity validation. aSyn = alpha-synuclein; ICC = immunocytochemistry; KO = knockout; MAP2 = microtubule associated protein 2; PBS = phosphate buffered saline; PFF = pre-formed fibril; WB = Western blot

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Extraction, Staining, Immunocytochemistry, Knock-Out, Saline, Western Blot

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: A selected panel of aSyn antibodies reveal the broad diversity of human pathology in the substantia nigra of LBDs. (A) An outline to show the epitopes of the aSyn antibody selection used for IHC studies on LBD tissues. Schematic created with BioRender.com (agreement no: NU23G6E7KK ). (B) Representative images from the substantia nigra of sporadic (PD, DLB) and familial ( SNCA H50Q) LBDs screened with the selection of aSyn non-modified and aSyn PTM antibodies. (C) Representative images from the cingulate cortex of sporadic (DLB) and familial ( SNCA G51D) LBDs screened with the selected aSyn PTM antibodies. aSyn = alpha-synuclein; DLB = dementia with Lewy bodies; IHC = immunohistochemistry; LBD = Lewy body disorder; PD = Parkinson’s disease; PTM = post-translational modification

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Selection, Modification, Immunohistochemistry

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Specificity validation of aSyn antibodies on post-mortem human tissues via IHC. The frontal cortices of PSP and CBD, and the hippocampi of AD, PiD and FTLD-TDP type C were stained using the selection of aSyn non-modified and PTM antibodies. No cross-reactivity was observed. Arrows indicate aSyn-positive structures detected on each tissue. AD = Alzheimer’s disease; aSyn = alpha-synuclein; CBD = corticobasal degeneration; ctx = cortex; FTLD-TDP/C = frontotemporal lobar degeneration of TAR DNA-binding protein 43 type C; hipp = hippocampus; IHC = immunohistochemistry; PiD = Pick’s disease; PSP = posterior supranuclear palsy

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Biomarker Discovery, Staining, Selection, Modification, Binding Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Development and validation of an expanded antibody toolset that captures alpha-synuclein pathological diversity in Lewy body diseases

doi: 10.1101/2022.05.26.493598

Figure Lengend Snippet: Application of the aSyn antibodies to the cellular and animal seeding models to profile the newly formed aSyn aggregates. WT hippocampal neurons were seeded with PFFs for 14 days, and the newly formed aggregates monitored by ICC using the mouse-reactive (A) non-modified aSyn and (B) aSyn PTM antibodies in parallel to aSyn pS129 antibodies BL 81A or AB MJF-R13. (C) The same type of screening was run in PFF-injected mouse amygdala tissues by IHC. The non-modified aSyn antibody signals overlapping with the aSyn pS129-positive aggregates are marked with an arrow. The punctate positivity shown by aSyn pY39, pY133 and pY136 antibodies in close proximity to aSyn pS129-positive aggregates are shown by arrowheads. Note the non-specific diffuse positivity revealed by the two monoclonal nY39 antibodies 5E1-G8 and 5E1-C10 in the WT hippocampal neurons are also revealed in the aSyn KO neurons using these two antibodies . aSyn = alpha-synuclein; ICC = immunocytochemistry; IHC – immunohistochemistry; KO = knockout; PFFs = pre-formed fibrils; PTM = post-translational modification; WT = wild-type

Article Snippet: We complemented our battery of antibodies against the aSyn PTMs with the previously generated in-house polyclonal antibodies against aSyn nY39 (LASH-EGT nY39), aSyn pY39 (LASH-EGT pY39), aSyn pS87 (LASH pS87), aSyn pY125 (LASH-EGT pY125), aSyn pS129 (LASH-EGT pS129), with the monoclonal aSyn pY39 antibody generated in collaboration with Biolegend (LASH-BL pY39) and with the commercially available aSyn pS129 antibody AB EP1536Y, which were also screened and validated in parallel to the novel monoclonal aSyn PTM antibodies.

Techniques: Modification, Injection, Immunocytochemistry, Immunohistochemistry, Knock-Out

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: CIB1 forms a complex with EVER1 and EVER2. (A) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*) and EVER2 (D362* or T150Mfs*3), and comparison with those in heterozygous carriers, positive controls (ctrl 1 and 2), P4 and P11, and patients with RHOH and MST1 deficiencies. (B) CIB1, EVER1, and EVER2 mRNA levels were assessed by RT-qPCR in controls ( n = 4), CIB1 m/m (P1–P6 and P12), EVER1 m/m, and EVER2 m/m ( n = 3 each) LCLs. Each symbol represents the mean of one cell line measured in three independent experiments. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison tests relative to healthy controls. (C) CIB1 protein levels in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. Asterisk indicates a nonspecific band. (D) CIB1, EVER1, and EVER2 mRNA levels were measured by RT-qPCR in LCLs derived from patients with loss-of-function mutations of EVER1 (D576*), EVER2 (T150Mfs*3), or CIB1 and a healthy control after reconstitution with WT EVER1 or EVER2 by retroviral transduction and stable selection. The data were first normalized against RNaseP as a housekeeping gene and then against an appropriate untransduced parental cell line by the ΔΔCt method. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the corresponding untransduced control. (B and D) ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (E) HEK293T cells were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either separately or together; 24 h after transfection, samples were subjected to immunoprecipitation with FLAG (Fl)- or HA-specific antibodies (+). Samples incubated with nonspecific IgG served as specificity controls. Western blots were performed to detect coimmunoprecipitated CIB1-HA and FLAG-EVER1 or FLAG-EVER2. The immunoprecipitation of EVER1/2 and CIB1 was confirmed by reincubation with antibodies specific for FLAG and HA, respectively. The presence of all proteins was checked by Western blotting of an input sample taken before immunoprecipitation. GAPDH served as a loading control. vec, vector. (F) Healthy control keratinocytes were transfected with plasmids encoding CIB1-HA, FLAG-EVER1, and FLAG-EVER2 either alone or in combination; 24 h after transfection, cells were subjected to immunofluorescence imaging with Alexa Fluor 568–HA and Alexa Fluor 488–FLAG antibody combinations. DAPI was used for counterstaining. Colocalization was assessed by calculating Pearson’s correlation coefficient with Imaris software. Bar, 13 µm. The results shown are representative of three independent experiments.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Derivative Assay, Comparison, Quantitative RT-PCR, Control, Retroviral, Transduction, Selection, Transfection, Immunoprecipitation, Incubation, Western Blot, Plasmid Preparation, Immunofluorescence, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: Analysis of zinc signaling/levels and NF- k B activation in the presence and absence of CIB1. (A) HEK293T cells were transfected with plasmids encoding CIB1, EVER1, and EVER2 either alone or in combination and with a 4×MRE-dependent EGFP reporter construct. After 24 h, cells were stimulated overnight with PMA/ionomycin (10 ng/ml and 50 ng/ml, respectively) or zinc sulfate (ZnSO 4 ; 100 µM). The next day, cells were stained with 1 µg/ml DAPI to exclude dead cells, and GFP fluorescence was determined with an LSRII flow cytometer. The RRR with the value for vector-transfected cells was set at 100%. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the appropriate vector-transfected control (ns, P > 0.05; *, P < 0.05; ***, P < 0.001; n = 3). (B) Flow cytometric quantification of absolute amounts of labile zinc in LCLs derived from healthy controls, EVER1-, EVER2-, or CIB1-deficient patients, or in keratinocytes from P14 with 1 µM FluoZin-3 as described by . Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison test relative to the healthy controls ( n = 3). (C) Kinetics of zinc flux in LCLs derived from healthy controls and EVER1-, EVER2-, or CIB1-deficient patients. Cells were loaded with 1 µM FluoZin-3 for 30 min. Fluorimetric measurement were performed on a Victor microplate reader. Baseline fluorescence was recorded every minute for 10 min. Cells were then loaded with 100 µM ZnSO 4 and recorded for 15 min. The specificity of the zinc signal was confirmed by adding the calcium-specific chelator BAPTA before the quenching of the signal with the zinc-specific chelator TPEN and recording for 20 min. No significant effect of genotype was detected in two-way repeat-measures ANOVA ( n = 3). (D) HEK293T cells were transfected with plasmids encoding CIB1-FLAG, FLAG-EVER1, and EVER2 either alone or in combination. Cells were stimulated with 50 ng/ml TNFα 6 h after transfection, incubated overnight, and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa and the noncanonical NF-κB component p100/p52. Expression of the constructs used for transfection was verified by incubation with a FLAG-specific antibody. GAPDH served as a loading control ( n = 3). (E) Primary keratinocytes from unrelated donors (controls 1 and 2), a healthy family member from kindred A1 carrying the mutation in a heterozygous state (A1.viii.2), and one patient each from kindreds A1 and C were stimulated with 10 ng/ml TNFα for 5, 10, or 20 min and then harvested and processed for Western blotting. Membranes were probed for the canonical NF-κB component (p)IκBa. CIB1 levels were assessed with a polyclonal antibody. GAPDH served as a loading control. vec, empty vector ( n = 3).

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Activation Assay, Transfection, Construct, Staining, Fluorescence, Flow Cytometry, Plasmid Preparation, Comparison, Control, Derivative Assay, Incubation, Western Blot, Expressing, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: The human CIB1–EVER1–EVER2 complex governs keratinocyte-intrinsic immunity to β-papillomaviruses

doi: 10.1084/jem.20170308

Figure Lengend Snippet: PLA and coimmunoprecipitation in HaCaT. (A) HaCaT cells were transfected with plasmids encoding FLAG-HPV5 E1, E2, E6, and E7, FLAG-HPV16 E1, E2, E5, E6, and E7, FLAG-HPV4 E8, FLAG–CRPV E8, and CIB1-HA alone or in combination. The day after transfection, samples were plated on microscopy slides, allowed to adhere, fixed in acetone, permeabilized, and subjected to Duolink PLAs with rabbit-HA– and mouse-FLAG–specific antibodies. Z stacks were acquired with a widefield microscope, and PLA-positive sites (defined as structures >0.35 µM 2 ) were scored with Imaris software for 15–50 cells per condition. These pooled results were obtained in two independent experiments. The orange bars indicate the mean. (B) HaCaT cells were transfected with plasmids encoding CIB1-HA and the FLAG-tagged HPV E ORFs scoring positive in the PLA in A. 1 d after transfection, samples were subjected to immunoprecipitation (IP) with FLAG-specific antibodies. Western blots were performed to detect coimmunoprecipitated HPV5 E1, HPV16 E2, E5, HPV4 E8, and CRPV E8. The immunoprecipitation of CIB1 was confirmed by reincubation with a FLAG-specific antibody. The presence of all proteins was checked by Western blotting analysis on an input sample taken before immunoprecipitation. GAPDH served as a loading control ( n = 3). vec, vector.

Article Snippet: The antibodies used were directed against the FLAG (anti-DDK; Origene) and HA epitopes (Sigma-Aldrich) and were used at a dilution of 1:500.

Techniques: Transfection, Microscopy, Software, Immunoprecipitation, Western Blot, Control, Plasmid Preparation