(A, B) FL Cys-Tau(2A) was labeled with Alexa488 and resolved by SEC (A), or was fibrillized in the presence of heparin, labeled with Alexa488, sonicated, and the assemblies resolved by SEC (B). The …
Data for Mi and Ms seeding activity in cells and in vitro.
(A) CD spectra of Mi and Ms were similar. (B) FCS Diffusion times for Mi, MS, dimer, trimer, and ~10 mer, and the cross-correlation for Mi, Ms, dimer, trimer, and ≥10 mer were determined after …
Data for CD and FCS of Mi and Ms.
SEC fidelity was tested by isolating Ms from fractions after fibril sonication. Remaining fractions were combined with Mi, and the mix was re-isolated by SEC. In Group 1, after the first isolation, …
Data for fidelity of SEC purification of assemblies.
(A–C) Heat-induced dissociation of assemblies. (A) The SEC fraction containing Ms (B5) was heated to 95°C for 3 hr and re-isolated by SEC prior to testing the FRET biosensor assay. No loss in …
Data for heat denaturation of assemblies.
Mi and Ms were incubated at 500 nM or with equivalent amounts (monomer equivalent) of dimer and trimer for various times prior to resolution by SEC. Assemblies were monitored by reading the …
Data for Ms self-assembly.
(A) Heparin treatment of FL WT tau was carried out for 15 min, 1 hr, or 4 hr. Samples were resolved by SEC, and fractions of various sizes were compared using the biosensor seeding assay. ‘Pre-SEC’ …
Data for heparin induced transition from Mi to Ms.
(A) Two different FL WT tau preparations were sonicated or not, and 1.5 µg protein was then resolved by SDS-PAGE and coomassie stain. Sonication induced a small degree of protein fragmentation. (B) …
Tau monomers were prepared as described, heated at 95°C for 0, 3 or 24 hr, reacted with DSS, proteolyzed and analyzed by mass spectrometry to define intramolecular crosslinks. Diagrams represent …
Summary of triplicate XL-MS datasets.
Summary of consensus XL-MS datasets.
Heat denaturation of Mi and Ms (fibril-derived and heparin treated for 0.25 hr, 1 hr) decreases the abundance of consensus crosslink pairs (A). Columns represent data after exposure to 95°C for 0 …
Tau from control and AD brains was immunoprecipitated and subjected to SEC. (A) SEC from control brain contained predominantly tau monomer. (B) SEC from AD brain contained a range of tau assembly …
Data characterization of patient samples.
Summary of patient-derived XL-MS datasets.
A single AD brain sample was homogenized using four different methods: (A) Dounce homogenization; (B) Pulse sonication; (C) Mechanical homogenization; (D) Mechanical homogenization followed by pulse …
XL-MS identified pairs were used as restraints in Rosetta to create structural models of discrete tau domains. (A) Schematic highlighting the region of the RD encoding structural differences between …
Models of Mi, Ms, control and AD conformations of tau.
Rosetta energy and radius of gyration for structural ensembles.
The ensembles are shown as a distribution of total energy of each model and radius of gyration for recombinant Mi (A), recombinant Ms (B), control brain-derived Mi (C) and AD-derived Ms (D). Figure …
(A) Mi and Ms were prepared as technical triplicates (N = 3), isolated by SEC, and passed through a 100kD filter immediately prior to exposure to trypsin for 1, 5, 10, 30, 60 and 120 min. Samples …
Summary of peptides and their abundance identified in the triplicate Mi and Ms proteolysis experiment.
Summary of ratiometric analysis of Mi and Ms peptide abundance.
The medians of the averaged (N = 3) kinetic profiles were compared as ratios for Mi and Ms. The data were compared to the mean of all ratios (red line) and standard deviation (dotted grey line). …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Cell line (HEK293) | Tau RD P301S FRET Biosensor | Produced by Diamond lab, also available from ATCC | ATCC CRL-3275 | |
biological sample (mouse) | Tau KO mouse | Jackson Laboratories | 7251 | |
biological sample (human) | Alzheimer's Disease brain, stage VI, frozen | Washington University in St. Louis | 62579, 62732, 61473 | |
biological sample (human) | Human normal brain | Washington University in St. Louis | 60974, 607, 64238 | |
antibody | HJ8.5 | Produced by Diamond lab | N/A | Monoclonal antibody against human tau |
antibody | Polyclonal Rabbit Anti-Human Tau | Dako, Denmark | A0024 | |
antibody | ECL Anti-rabbit IgG Horseradish Peroxidase F(ab) fragment | GE Healthcare | NA9340V | |
recombinant protein | Human tau 2N4R (Full Length WT-tau) | Produced by Diamond lab | N/A | MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L |
recombinant protein | Tau (2A) | Produced by Diamond lab | N/A | MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK AGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KAGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L |
recombinant protein | Cys-Tau (2A) | Produced by Diamond lab | N/A | MAEPRQEFEV MEDHACGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSP GSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK AGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KAGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVS GDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L |
commercial assay or kit | micro BCA Protein Assay Kit | Thermo Scientific | 23235 | |
chemical compound | Heparin sodium salt from porcine intestinal mucosa | Sigma | H4784 |