Ab75714

Immunoblotting was performed as previously described [28 (link)]. Antibodies and dilutions used in this study include: anti-TrkB (1:1000; # ab18987, Abcam), anti-phospho-TrkB (phTrkB Y817; 1:1000; # ab81288, Abcam), anti-synapsin I (1:1000; # ab64581, Abcam), anti-PSD-95 (1:1000; # ab12093, Abcam), anti-tau (1:1000; # ab75714, Abcam), anti-phospho-tau (phospho T181; 1:1000; # ab75679, Abcam), anti-phospho-tau (phospho S262; 1:1000; # ab131354, Abcam), anti-phospho-tau (phospho S396; 1:1000; # ab109390, Abcam), and anti-β-actin (1:1000; # ab1801, Abcam). Quantitative densitometric analyses were performed on digitized images of immunoblots in the ImageJ software (NIH, USA).

anti-CD9 (Abcam, ab92726, 1:1,000), anti-CD81 (clone B-11, sc-166029, 1:1,000, Santa Cruz), anti-EphrinB1 (ECD epitope, AF473, R&D Systems, 1:500), anti-EphrinB1 (ICD epitope, LifeSpan BioSciences, LS-C108001, 1:500), anti-NGF (Abcam, ab49205, 1:500), anti-β-III Tubulin (ab18207, Abcam, 1:1000), anti-GAPDH (ThermoFisher, AM4300, 1:5,000), anti-β actin (1:500, A2228, Sigma), anti-Tau (1:500, Abcam, ab-75714), anti-Phosphorylated Erk1/2 (1:500, #9106 s, Cell Signaling), anti-Erk1/2 (1:500, ABS44, Millipore), anti-TRPV1 (Novus, NB100-98897, 1:500). HRP- coupled secondary antibodies purchased from Thermo-Fisher. Citations for these validated antibodies are listed in Supplementary Table 9.

For ICC performed on iPSC-derived spinal motor neurons, neurons were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 for another 15 min. The cells were then blocked with 5% donkey serum at 25 °C for 1 h before being incubated with primary antibodies. The primary antibodies used were anti-PITX2 (1:200, H00005308-M01, Novus Biologicals), anti-Tau (1:500, ab75714, Abcam), anti-Bassoon (1:200, ab110426, Abcam), anti-MAP2 (1:1000, ab5392, Abcam), and anti-Homer1 (1:200, ab97593, Abcam). Secondary antibodies used in this study were Alexa Fluor 488 Goat anti-Chicken IgY H&L (1:500, ab150169, Abcam), Alexa Fluor 594 Donkey anti-Mouse IgG (H + L) (1:500, A-21203, Thermo Fisher Scientific), and Alexa Fluor 647 Donkey anti-Rabbit IgG (H + L) (1:500, A-31573, Thermo Fisher Scientific). The number of Bassoon or Homer1 puncta was counted alongside each neurite of 50 μm in length. The cell nuclei were stained with Hoechst 33342 (1:400, H-1399, Thermo Fisher Scientific). Cell images were acquired on an Olympus IX-81 FV1000 confocal microscope (Olympus) using a 63x water immersion objective lens and Olympus Fluoview software (Version 4.2a). Only representative images are shown.

Immunofluorescence assays were performed with frozen sections of frontal cortices from human and mouse brains. The sections were fixed in chilled methanol followed by blocking in blocking buffer (5% BSA and 5% normal goat serum in 1X PBS supplemented with 0.25% Triton X-100 (PBST)) for 2 h at RT. Sections were then incubated in primary antibodies diluted in 5% BSA in PBST for overnight at 4 °C, including rabbit anti-T18 (1:500), chicken anti-Total Tau (1:1000, Abcam; ab75714), rabbit anti-ubiquitin (linkage-specific K48) (1:500, Abcam; ab140601), rabbit anti-ubiquitin (linkage-specific K63) (1:500, Abcam; ab179434), mouse anti-ubiquitinylated protein, clone FK2 (1:500, Sigma-Aldrich; 04–263), mouse anti-βIII-tubulin (1:1000, Abcam; ab78078), chicken anti-GFAP (1:1000, Abcam; ab4674), or guinea pig anti-IBA1 (1:500, Synaptic Systems; 234,004). After washing three times with 1X PBS (10 min each), sections were incubated in Alexa-conjugated antibodies (1:700, Invitrogen) diluted in 5% BSA in PBST for 2 h at RT. Slides were washed and mounted using Prolong Gold antifade reagent with DAPI (Invitrogen, P36935).