Lc6070

For in vitro cleavage assays, 1 µg of recombinant full-length human α-syn (Abcam #ab51189), TDP-43 (R&D #AP-190) or 4N2R tau (rPeptide #T-1001–1) was incubated with or without 1 µM of each protease. Proteases requiring pre-activation were performed, as mentioned in Table S2. The following buffers were used as indicated: 100 mM sodium citrate pH 3.4, 50 mM sodium acetate pH 4.5 or 5.5 or 100 mM phosphate buffer saline (PBS) pH 7.4. Also, 1 mM EDTA and 2 mM DTT were used and each reaction was performed over 1 h at 37ºC. The assay was performed in a total volume of 19.5 µl. Protease activity was stopped by adding 7.5 µl of NuPAGE 4X LDS (Fisher Scientific #NP0007) and 3 µl of 10X reducing agent (i.e., 50 µM) (Fisher #NP0009). Samples were then immediately denatured for 10 min at 80ºC. All samples were run on precast NOVEX 4–12% Bis–Tris gels (Fisher #NP0321PK2) using MES buffer (Fisher #NP0002). The gel was then either fixed in 40% ethanol and 10% acetic acid for silver stain or transferred onto nitrocellulose membranes for Western blotting. Silver staining was performed according to the manufacturer’s instructions (Thermo Fisher #LC6070).

Control and TAP-CrkL samples were separated by SDS-PAGE and visualized by silver staining (LC6070; Invitrogen). Each lane was cut into 17 equal individual slices without regard to the staining pattern. The samples were destained, reduced with 20 mM dithiothreitol (DTT), and alkylated with 50 mM iodoacetamide. The samples were then digested overnight with 0.1 μg trypsin per gel slice. Tryptic peptides were extracted, dried under a vacuum, and then resuspended in 12 μl 0.1% formic acid. Eight microliters of each sample was loaded onto a 75-μm by 12-cm column self-packed with 3 μm ReproSil-Pur C₁₈-AQ beads (Dr. Maisch GmbH), eluted with a gradient of 2 to 40% acetonitrile in 0.1% formic acid over 50 min at 300 nl/min, and analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Proteins were identified using the Andromeda search engine (MaxQuant version 1.2.2.5) with cysteine carbamidomethylation specified as a fixed modification and methionine oxidation as a variable modification to search the Swiss-Prot database. Relative quantities of the proteins were determined using the iBAQ feature of MaxQuant. The entire data set is included as Data Set S1 in the supplemental material, and the raw data are hosted at ftp://massive.ucsd.edu/MSV000079311/.

FXII activation by Aβ40 was measured by a chromogenic assay [22 (link)], monitoring the conversion of the chromogenic substrate for 3 h at room temperature (RT) using a microplate reader. To measure its degradation by FVIIa, Aβ40 was incubated with or without FVIIa plus cofactor TF in PBS in a total volume of 100 μl containing 5 mM CaCl₂ for 1 or 6 h at RT. TF was solubilized in 10 mM CHAPS, which was diluted 10-fold in the final assay. FVIIa and TF were also incubated without Aβ40, as a control. Each sample at an equal volume was subjected to SDS-PAGE and stained by silver using a kit from Invitrogen (LC6070).

Reconstitution of human telomerase with the DNA primer and TPT was carried out previously, as described in Sekne et al. ^{36 (link)}. Briefly, telomerase lysates were subjected to the oligo purification as described in Telomerase expression and purification. The eluate from the oligo purification was added to the pre-washed MagStrepXT resin (IBA LifeSciences, Cat# 2-4090-002) and incubated overnight at 4 °C. The resin was washed three times with wash buffer and then incubated with 2 μM telomerase DNA primer (T₂AG₃)₅ for 30 min at room temperature. After washing, purified TPT was added to the resin at a final concentration of 0.15 mg ml⁻¹ and incubated for 1 hour at 4 °C. The complex was eluted by incubation in biotin elution buffer (100 mM HEPES NaOH pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM biotin, 2 mM MgCl₂, 10% glycerol, 0.1% IGEPAL CA-630, 0.2 mM PMSF and 1 mM DTT) for 1 hour at 4 °C. Fractions were analyzed on the SDS-PAGE gel followed by silver staining (Invitrogen, Cat# LC6070). The presence of the telomeric DNA substrate in the final elution was confirmed by telomerase activity assays.