Anti 6x his tag

Samples were separated by SDS-PAGE gel, and transferred to PVDF membranes using the Trans-Blot Turbo Transfer system (Bio-Rad, Hercules, CA, USA), and probed with antibodies Anti-6X His-tag (ab1187; Abcam, Cambridge, UK). Blots were developed by ECL (Bio-Rad, Hercules, CA, USA).

To perform cleavage assays the protein containing media from transient transfections was mixed and incubated for 1–7 hr at 25°C. Reaction volumes ranged from 30–51 μl containing: 10–30 μl Sog-Myc with 0–10 μl Tld-HA proteins and a combination of 0–10 μl Tsg-V5, 0–10 μl VkgC-FLAG/Dcg1C-FLAG (co-transfected) and 1–20 nM recombinant Dpp (rDpp). Recombinant Dpp was purchased from R&D systems (Minneapolis, MN) and reconstituted in 4 mM HCl. Total reaction volume was kept constant by the addition of mock transfected cell medium. When complete, reactions were stopped with sample buffer (2.5% glycerol, 12.5% SDS, 20% β-mercaptoethanol, 24 mM Tris/HCl) and analysed by Western blot. Cleavage assays using the zipper fusion proteins were performed by co-transfecting the appropriate plasmids, as described above.
Antibodies used as follows; 1:500 anti-Myc antibody (clone 4A6, Millipore, Darmstadt, Germany), 1:1000 anti-V5 tag (monoclonal AV5-PKI, AbCam, Cambridge, UK), 1:1000 anti-6xHis tag (AbCam), 1:1000 anti-HA (Roche, Basel, Switzerland), 1:1000 anti-flagM2 (SIGMA). Blots were visualised using either ECL reagent (GE Healthcare, Buckinghamshire, UK) or West Dura Supersignal (Thermo Scientific, Waltham, MA) with the ChemiDoc MP System (BioRad, Hercules, CA) or exposed using Biomax film (Kodak, Rochester, NY). For quantification ImageJ Version 10.2 or ImageLab (BioRad) was used.

FFPE tissue was dewaxed in xylene, rehydrated in graded alcohols and then blocked with 10% H₂O₂ for 1 h at RT in the dark. Antigen retrieval was performed via microwave heating with citrate buffer pH-6 (tau5, AT180, anti-6X His tag), immersion in 80% formic acid for 1 h (4G8) or no treatment (AT8, MC1). Then sections were blocked with 10% normal goat serum in TBS-0.1% Triton X-100 for 1 h at RT, incubated with tau5 (1:200), anti-beta amyloid clone 4G8 (Biolegend, 1:2000), AT8 (Innogenetics, 1:500), AT180 (ThermoFisher Scientific, 1:1000), MC1 (kindly provided by Peter Davies, The Feinstein Institute for Medical Research, Manhasset, NY, 1:100), or anti-6X His tag (Abcam, 1:200) at 4 °C overnight, incubated with biotinylated goat anti-mouse/anti-rabbit IgG secondary antibody (Jackson Immunoresearch) for 1 h at RT, washed in TBS-0.1% Triton X-100, incubated with VectaStain ABC reagent (Vector Labs) for 1 h at RT, treated with 3,3′-Diaminobenzidine (DAB, Sigma) for 3 min, counterstained with hematoxylin, dehydrated in graded alcohols and xylene and mounted with DPX mounting reagent.
PK treatment was performed as an additional step after antigen retrieval by incubating slides in 50 µg/ml PK (Sigma) 10 mM Tris HCl pH 7.8, 100 mM NaCl, 0.1% NP-40 at 37 °C for the stated times (0 s, 10 s, 1 min and 2 min).