Complete mini protease inhibitors cocktail

DISC precipitation was performed using biotin-labeled recombinant TRAIL (biotin-TRAIL)^{34 (link)}. Cells were treated with bio-TRAIL for the times indicated in the figure legends. DISC formation was then stopped and unbound TRAIL was removed by washing the cells three times in ice-cold PBS. Cells were lysed in 3 mL of lysis buffer (30 mM Tris/HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% of sodium deoxycholate) containing Complete mini protease inhibitors cocktail (Roche Molecular Biochemicals)) for 30 min on ice followed by centrifugation at 13 K for 30 min at 4 °C. To provide an unstimulated receptor control, biotin-TRAIL was added to lysates from untreated cells. The TRAIL DISC was collected from the lysates with 30 μL of streptavidin-agarose beads after overnight incubation at 4 °C. Beads were collected by centrifugation, washed six times with lysis buffer and receptor complexes were eluted with 30 μL of sample buffer. Proteins were analyzed by western blotting.

The left hemisphere of each brain was homogenized in 7 volumes of 10 mM Tris-HCl, pH 7.4, added with cOmplete Mini Protease Inhibitors cocktail (Roche), sonicated for 1 min using an ultrasonic homogenizer (SONOPULS) and centrifuged at 100,000 xg for 1 h at 4 °C. The supernatant was saved as the soluble fraction; the pellet was extracted in 70% formic acid and neutralized with 20 volumes of 1 M Tris (insoluble fraction).
Aβ40, Aβ42 and aggregated Aβ were measured in both soluble and insoluble fractions by ELISA (Invitrogen). Each experiment was performed in triplicate. The analysis of APP processing followed the protocol described above. After centrifugation, the supernatant was collected as the SDS-soluble fraction, which was analyzed by western blot with the A8717 antibody (Sigma). Signal intensity of the bands was measured by Quantity One (BioRad).

The western blotting experiments were performed as previously described [26 (link)]. The following antibodies were used: mouse monoclonal anti-GAPDH (1:4 000 dilution, G8795, Sigma-Aldrich), anti-HYAL1 (1:1 000 dilution, 1D10, produced by hybridoma cells generously provided by B. Triggs-Raine, University of Manitoba, Winnipeg, Canada) and anti-cathepsin K (1:1 000 dilution, MAB3324, Millipore), as well as goat polyclonal anti-TRAP (1:1 000 dilution, SC-30833, Santa Cruz Biotechnology). When conditioned media were prepared, the cells were cultured for 5 h in serum-free medium prior to lysis of the cells in PBS—Triton X-100 1% supplemented with protease inhibitors (cOmplete, mini protease inhibitors cocktail, Roche). When indicated, cells were incubated for 24 h with either 15 mg/mL of mannan (Sigma-Aldrich) or 5 mM of Man-6-P (Sigma-Aldrich). In Fig 5C, cell extracts and conditioned media were treated with PNGase F (New England Biolabs) according to the manufacturer's instructions. Of note, the specificity of the anti-HYAL1 antibody was validated by an absence of signal in osteoclasts differentiated from BMM of Hyal1 -/- mice (S1A Fig). HYAL1 signals were quantified using the ImageJ (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997–2015).