Additional file 1 of Robust α-synuclein pathology in select brainstem neuronal populations is a potential instigator of multiple system atrophy
Creators: name: Hass, Ethan W.
name: Sorrentino, Zachary A.
name: Lloyd, Grace M.
name: McFarland, Nikolaus R.
name: Prokop, Stefan
name: Giasson, Benoit I.
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Neuroscience
subject: Pharmacology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: FOS: Earth and related environmental sciences
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Computational Biology

description: Additional file 1: Supplementary Fig. 1. Location of the epitopes for the new αSyn antibodies. The amino acid sequences of human and mouse αSyn, as well as human γSyn and human βsyn, are shown with points of non-homology to human αSyn highlighted. The three regions of αSyn (N-terminus, NAC region and C-terminus) are labeled and displayed above the sequences. The epitopes of the five novel C-terminal αSyn antibodies were determined based on the peptide used for immunization and immunoblot analysis using carboxy--truncated recombinant αSyn protein (see Additional file 1: Supplemental Fig. 5). Supplementary Fig. 2. IHC staining of tissue sections from LBD patients in the substantia nigra, cingulate cortex and amygdala with the various αSyn antibodies labeling Lewy bodies and Lewy neurites. Tissue sections were stained with the αSyn antibodies indicated in the top of each column. All sections were counterstained with hematoxylin. Scale bar = 100 μm. Supplementary Fig. 3. IHC staining of cerebellum tissue sections from an MSA patient with the various αSyn antibodies labeling GCIs. Tissue sections were stained with the αSyn antibodies indicated in the top left corner. All sections were counterstained with hematoxylin. Scale bar = 100 μm. Supplementary Fig. 4. IHC staining with αSyn antibody 5H12 in a control patient. Tissue sections from a the cerebellum, b pons and c medulla oblongata showing the paucity of pathological inclusions. Sections were counterstained with hematoxylin. Scale bar = 300 μm. Supplementary Fig. 5. Western blot analysis to refine the epitope map and characterize the specificity of the new αSyn antibodies. Immunoblot analysis using recombinant human γSyn, human βSyn, mouse (m) αSyn, human (h) αSyn and a series of carboxy-truncated human αSyn (1–129, 1–125, 1–122, 1–119 and 1–115) proteins. 210 ng of each protein was loaded per lane and membranes were probed with the antibodies labeled above. The relative mobility of molecular mass markers is indicated on the left of each blot. Supplementary Fig. 6. Schematic for biochemical fractionation of human brain tissue. Horizontal arrows indicate the solutions added to either the homogenized tissue or pellet resulting from the previous step. From top to bottom, the collected fractions were high salt, HS/Triton X-100 fraction radioimmuno-precipitation assay (RIPA) and SDS/urea fractions. (PDF 11290 kb)