Fluorescent streptavidin

Cell pellets were resuspended in 200 μl lysis buffer (8 M urea, 50 mM Tris–pH 7.5, protease inhibitors (Merck)). From there on, processing, SDS–PAGE separation, Western blotting and fluorescent‐based imaging was done as described in Eisenberg‐Bord et al (2021 (link)), with the exception of the HA blot in Fig EV1C. Here, the SDS–PAGE gel was blotted onto PVDF membrane (Millipore) by wet transfer, and imaging was done using X‐ray film (FujiFilm) to detect signal from HRP‐conjugated anti‐mouse secondary antibody (1:7,500, Jackson ImmunoResearch, #111‐035‐003) incubated with ECL substrate (Thermo Scientific). The following antibodies were used for Western blot: anti‐HA (1:1,000, BioLegend, #901502), anti‐Myc (1:3,000, Abcam, #ab9106), anti‐Histone H3 (1:5,000, Abcam, #ab1791), anti‐Sec61 (1:5,000, a kind gift from Matthias Seedorf of Heidelberg University and Marius Lemberg of the University of Cologne), anti‐Actin (1:2,000, Abcam, #ab8224), goat anti‐rabbit IgG H&L 800CW (1:7,500, Abcam, #ab216773) and goat anti‐mouse IgG H&L 680RD (1:7,500, Abcam, #ab216776). Membranes were incubated for 1 h at RT with fluorescent streptavidin (1:10,000, Invitrogen, #S11378) diluted in 2% (w/v) BSA/PBS containing 0.01% NaN₃ to detect biotinylated proteins.

Following fixation in 10% buffered formalin, half of the P301L MAPT mutant patient brain was sliced in the coronal plane and frontal cortex used for histological examination. The other half of the brain was sliced in the coronal plane, flash frozen and stored at −80°C. Paraffin-embedded sections (7 μm) from frontal cortex were deparaffinized, rehydrated and endogenous peroxidase activity blocked with methanol/0.3% H₂O₂. Immunohistochemistry was performed as previously reported (Spillantini et al., 1998a (link)). Briefly sections were incubated overnight at 4°C with primary antibody, washed, and incubated at room temperature for 2 h with biotinylated secondary antibody (1:250, Vector Laboratories), before incubation for 1 h with Avidin-DH (1:100, Vectastain Elite Kit, Vector laboratories). Staining was developed using 3,3 diaminobenzidine/H₂O₂ and sections counterstained with cresyl violet before mounting in DPX (Sigma-Aldrich). For immunofluorescence, following incubation with biotinylated secondary antibody, sections were incubated with fluorescent streptavidin (Alexa, Invitrogen). Paraffin sections from cerebral cortex of two control subjects (Table 1) were obtained from the Cambridge Brain Bank and processed as indicated above.