Fastprep machine

The α-mannosidase and α-glucosidase activities were measured using the fluorogenic substrates 4-methylumbellyferyl-α-D-mannopyranoside (Sigma) and 4-methylumbellyferyl-α-D-glucopyranoside (Sigma), respectively. Cells were harvested by centrifugation, broken with glass beads in a FastPrep machine (Thermo Scientific), and the homogenate centrifuged at 21,000 × g and 4°C for 10 min. The supernatant was saved and used to quantify enzyme activity as reported (Mora-Montes et al., 2004 (link); Robledo-Ortiz et al., 2012 (link)). Aliquots containing 100 μg protein were resuspended in 10 mM phosphate buffer, pH 7.0, in a total volume of 200 μL. Then, 40 μM of either 4-methylumbellyferyl-α-D-mannopyranoside or 4-methylumbellyferyl-α-D-glucopyranoside were added and the reaction incubated at 37°C for 30 min. The reaction was stopped by addition of 3.3 mL 50 mM glycine-NaOH, pH 11.0, and fluorescence of the released 4-methylumbellyferone (MU) was measured in a Perkin-Elmer LS-5B luminescence spectrofluorometer, with excitation and emission set at 350 nm and 440 nm, respectively. Total enzyme activity was expressed as nmoles of MU min^-1 total protein^-1. In assays to inhibit mannosyl-oligosaccharide glucosidase, 10 μM castanospermine (Sigma) was added prior the incubation step at 37°C (Lopes-Bezerra et al., 2015 (link)).

Logarithmically growing cells were harvested and frozen at −80°C. Pellets were resuspended in the same volume of HB2 buffer (50 mM HEPES/KOH at pH 7.5, 140 mM NaCl, 15 mM EGTA, 15 mM MgCl₂, 0.1% NP-40, 0.5 mM Na₃VO₄) containing protease inhibitors (1 mM dithiothreitol, 1 mM PMSF, 0.1% Protein inhibitor cocktail set III (Sigma), 0.1 ng/ml MG132 (Sigma), 10 U/ml TURBO DNase (Ambion)). Cells were broken using a Fast Prep machine (Thermo), briefly sonicated using the Biorupter and centrifuged to harvest the chromatin containing cell extract. Monoclonal anti-Flag M2 antibody (Sigma), anti-Myc 9E11 (Cell Signalling), anti-PK antibody (AbD Serotec/Bio-Rad) and anti-HA antibody (Covance) were used for pull down and detection of FLAG, Myc, PK and HA tagged proteins respectively. Anti-Cdc2 antibody (Santa Cruz) was used as a control. For protein complex immunoprecipitation, antibodies were conjugated with mouse IgG-coated Dynabeads (Life Technologies).

Frozen elk obex samples were partially thawed and approximately 200 mg of tissue was collected from the interior of the obex sample, placed into a 2 ml tube containing silica beads and 180 μl of sPMCA buffer #1 (150 mM NaCl, 4 mM EDTA, in PBS) was added. Tissues were homogenized using a FastPrep machine (Thermo Scientific) as outlined in Meyerett et al.46 (link). The clarified 10% homogenate supernatant was removed and stored at −80°C.
sPMCA substrate consisted of 10% mouse normal brain homogenate (NBH) prepared in a prion-free room from Tg5037 mice expressing cervid PrP^C as previously described46 (link).