(A) Scheme of NanoBiT-based intramolecular (conformational) and intermolecular (interaction) biosensors. (B) Biosensors were developed for three tau forms: full-length wild-type tau (WT-Tau) …
Unedited blot of Figure 1E.
(A) Thioflavin T (ThT) signal of recombinant tau, K18, and Aß proteins (10 µM) prepared as monomeric (m), oligomeric (o), or aggregated forms (agg). (B–C) K18(P301S) interaction signal in biosensor …
(A) Saturation curve of the intermolecular WT-Tau interaction sensor in HEK293T cells expressing a fixed amount of LgBit-Tau (250 ng of a transfected vector) and increasing amounts of SmBit-Tau or …
Unedited blot of Figure 2A.
Unedited blot of Figure 2E.
Unedited blot of Figure 2K.
Unedited blot of Figure 2L.
Unedited blot of Figure 2M.
(A) Thioflavin T (ThT) aggregation kinetics assay of recombinant full-length WT tau (10 µM) in the absence or presence of aggregation inducer (heparin 1:1) and in the absence or presence of …
(A) WT-Tau interaction biosensor signal in cells treated with monomeric (m) or aggregated (agg) forms of recombinant K18 or tau; data are expressed as mean ± SEM of five independent experiments. (B) …
(A) Biosensor signal in cells expressing K18(P301L) interaction biosensor or the non-related LgBiT-K18(P301L)+SmBiT Halo biosensor interaction pair following treatment with brain lysates obtained …
(A–B) Tau(P301L) interaction (A) and conformation (B) biosensor signal in cells treated or not with forskolin (10 µM, 24 hr); data are expressed as mean ± SEM of four (A) or five (B) independent …
Unedited blot of Figure 4A.
Unedited blot of Figure 4B.
(A) Thioflavin T (ThT) aggregation kinetics assay of recombinant full-length tau(P301L) and LgBit-Tau(P301L) proteins (10 µM), in the presence of aggregation inducer (heparin 1:1). (B–C) Tau(P301L) …
(A–B) Determination of the assay Z-factor by collecting biosensor signals for K18(P301L) (A) or Tau(P301L) (B) interaction biosensor cells in the presence of brain lysates from wild-type (WT) (red, …
(A) Cell viability assessed by MTT test in HEK293T cells incubated with vehicle (DMSO 0.1%) or the corresponding compounds (10 µM, 24 h); Triton 20% was used as a positive control of the assay; data …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
gene (Homo sapiens) | MAPT | GenBank | NCBI Entrez Gene: 4137 | |
strain, strain background (Escherichia coli) | BL21(DE3) | Sigma-Aldrich | CMC0016 | Electrocompetent cells |
cell line (Homo sapiens) | Human Embryonic Kidney (HEK) 293 | Sigma-Aldrich | RRID: CVCL_0063 | |
cell line (Homo sapiens) | Human neuroblastoma SH-SY5Y cells | Sigma-Aldrich | RRID:CVCL_0019 | |
transfected construct (Tag constrcut) | NanoBiT system (SmBit and LgBit) | Promega | ||
transfected constructo (Homo sapiens) | LgBit-HA-K18(P301L) | This paper | see Methods | |
transfected construct (Homo sapiens) | SmBit-HA-K18(P301L) | This paper | see Methods | |
transfected construct (Homo sapiens) | LgBit-HA-Tau(P301L) | This paper | see Methods | |
transfected constructo (Homo sapiens) | SmBit-HA-Tau(P301L) | This paper | see Methods | |
transfected constructo (Homo sapiens) | LgBit-HA-Tau | This paper | see Methods | |
transfected constructo (Homo sapiens) | SmBit-HA-Tau | This paper | see Methods | |
antibody | anti-HA antibody (rabbit monoclonal) | Cell Signaling | Cat# 3724 S | IF(1:500), WB (1:1000) |
antibody | anti-HA antibody (mouse monoclonal) | Biolegend | Cat#. 901514 | IF(1:500) |
antibody | anti-tubulin (rat polyclonal) | Millipore | Cat#: MAB1864 | IF(1:200) |
antibody | anti-phospho tau antibody AT8 (Mouse monoclonal) | Thermofisher | MN1020; | IF(1:200), WB (1:500) |
antibody | anti-aggregated tau MC-1 antibody (Mouse monoclonal), conformation-specific anti-tau antibody | PMID:9349554 | IF(1:500) | |
antibody | IRDye 800CW anti-Rabbit IgG (Goat polyclonal) Secondary Antibody | LI-COR Biosciences - GmbH | Cat.# 926–32211 | WB (1:10,000) |
antibody | IRDye 680RD anti-Mouse IgG (Goat polyclonal) Secondary Antibody | LI-COR Biosciences - GmbH | Cat.# 926–68070 | WB (1:10,000) |
commercial assay or kit | nanoluciferase substrate Nano-Glo Live Cell | Promega | Cat.# N2012 | |
commercial assay or kit | Duolink PLA | Sigma-Aldrich | Cat.# DUO92101 | |
chemical compound, drug | thioflavin S | Sigma Aldrich | Cat.# T1892 | |
chemical compound, drug | thioflavin T | Sigma Aldrich | Cat.# T3516 | |
software, algorithm | Image J | https://doi.org/10.1038/nmeth.2089 | RRID: SCR_003070 https://imagej.nih.gov/ij/download.html | |
software, algorithm | GraphPad Prism 6 | GraphPad Software Inc | RRID:SCR_002798; https://www.graphpad.com/scientificsoftware/-prism/ | |
other | EnVision Plate Reader | PerkinElmer | see Methods |