Complete ultra protease inhibitor

Tissues were lysed with Thermo Scientific Pierce T-PER Tissue Protein Extraction Reagent (Thermo Scientific., St. Louis, MO, USA) containing cOmplete ULTRA Protease Inhibitor and PhosSTOP Phosphatase Inhibitor Cocktails (Roche, Branchburg, NJ, USA). Proteins were separated by 4 ~ 12% SDS-PAGE gel electrophoresis and transferred to an Immun-Blot PVDF Membrane. The membrane was then incubated with specific primary antibodies, i.e., rabbit anti-TTR antibody (1:1000 dilution; ABBIOTEC., San Diego, CA, USA) or rabbit anti-NPY antibody (1:1000 dilution; ImmunoStar., Hudson, USA), followed by incubation with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Sigma-Aldrich), and detected by the SuperSignal West Pico Chemiluminescent Substrate Kit (Thermo Scientific). Anti-β-actin antibody was used for loading controls.

The strain carrying the constitutively expressed mCherry V1 was grown overnight in 50 mL of LB media supplemented with 100-µg/mL ampicillin at 37°C, 225 rpm. Cells were harvested by centrifugation (5,000 rpm, 5 min) and resuspended in lysis buffer (50 mM Tris-HCl, 50 mM NaCl, 0.05% Triton X-100, pH 8.0) supplemented with 1 tablet EDTA-free cOmplete ULTRA protease inhibitor (Roche). Cell debris were eliminated via centrifugation (7,500 rpm, 15 min), and the soluble fraction was collected.

Total protein was extracted from cell samples using RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) supplemented with 1× complete ULTRA protease inhibitor (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Proteins were resolved by 7.5%, 10%, or 12% bis-tris polyacrylamide gels and were transferred to polyvinylidene fluoride membranes and then blocked and probed with the appropriate antibodies overnight at 4 °C. Finally, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h and visualized with an enhanced chemiluminescence detection system. Detailed information of the antibodies is found in Supplementary Table S3.

α-PrP^23–231 and β-PrP^23–231 at pH 6.0 were digested with varying concentrations (0.005–50 μg/ml) of proteinase K (PK; British Drug Houses) at 37 °C for 1 h and diluted to 1 mg/ml in 10 mm sodium acetate, 10 mm Tris acetate, pH 6.0. Digestion was terminated by the addition of Complete ULTRA Protease Inhibitor (Roche Applied Science). Samples were heated to 100 °C for 5 min in SDS loading buffer before electrophoresis on 16% Tris glycine polyacrylamide gels (Novex). Gels were Coomassie stained.

The biotin switch assay was performed following a method applied for cysteine modification detection of yeast ER Hsp70 Kar2 with some modifications (27 (link), 59 (link)). The supernatant of the cell lysis (3 ml) was mixed with 12 ml of urea-containing cysteine modification buffer (CMBU; 0.1 m HEPES-NaOH, pH 7.4, 1% SDS, 1 mm EDTA, 8 m urea) with cOmplete ULTRA protease inhibitor (Roche Applied Science) and 0.1 m NEM. Samples were placed for 30 min at room temperature (RT). Proteins were precipitated with 10% TCA on ice for 30 min. The pellet was collected by centrifugation and washed once with 5% TCA and twice with 70% acetone and dissolved in 300 μl of CMBU. Then 6 ml of Grx reduction buffer (0.1 m Tris-HCl, pH 8.0 containing 1 mm EDTA, 0.5 mm GSH, 1 mm NADPH (Sigma–Aldrich), 0.25 units/ml GSH reductase (Sigma–Aldrich) and 60 μg of purified E. coli Grx3 C14S/C65Y (27 (link), 33 (link), 38 (link), 44 (link), 64 (link)) or Grx3 C11S/C14S/C65Y (27 (link))) was added, and samples were incubated for 15 min at 37 °C. After reduction, samples were quenched with TCA, and protein precipitation was carried out as above. The pellet was dissolved in 300 μl of CMBU with 0.2 mm biotin-maleimide (Sigma–Aldrich) and was placed for 30 min at RT and then protein precipitation was carried out as above. The pellet was dissolved in 100 μl of CMBU.