F60 sonic dismembrator

For normal brain homogenate sPMCA, brain homogenates were prepared at 10% (w/v) in conversion buffer (PBS containing 1% (v/v) Triton X-100 and cOmplete protease inhibitor cocktail (Roche)) from healthy C57BL/6 mice, as described by Castilla et al. [5 (link)]. Homogenates were clarified by brief sonication (F60 Sonic Dismembrator (Fisher Scientific), three 5-s pulses at ~1.8 amplitude), followed by centrifugation at 500 rcf for 15 min. Clarified substrates were then seeded with 1/10^th volume of purified PrP^Sc samples, and sPMCA carried out as described above, with 20-s sonication pulses every 30 min.
For deglycosylated PrP^C sPMCA, diglycosylated native PrP^C was first purified from normal mouse brain and then fully deglycosylated by treatment with PNGase F (New England Biolabs) as described [63 (link)]. Deglycosylated PrP^C sPMCA reactions [63 (link)] were seeded with 5% (v/v) of recombinant or brain-derived PrP^Sc and sonicated as described above for normal brain homogenate sPMCA.

Cells were scraped in 1× lysis buffer from Cell Signaling Technology (catalog no. 9803) with protease and phosphatase inhibitors and 0.5% SDS, followed by sonication with F60 Sonic Dismembrator (Fisher Scientific). The amount of protein in each sample was quantified using Pierce BCA Protein Assay Kit. All samples were diluted to the same concentration. For western blot analysis, samples were boiled at 95 °C for 5 min and 20 μg of total protein was loaded on Criterion™ TGX™ precast gels (BioRad Laboratories). Gels were run for 1 h at 150 V followed by transferring proteins onto a nitrocellulose membrane using an iBlot system (Invitrogen). Five percent non-fat dried milk in tris-buffered saline, tween 20 was used to block the membranes for 1 h. After blocking, primary antibodies against GCLC (Abcam), NQO1 (Abcam), HO1 (Abcam), TXNRD1 (Abcam), ACTB (MP Biomedical), Phospho-IκBα (Abcam), and GAPDH (Abcam) were incubated overnight at 4 °C, followed by washing with 1× tris-buffered saline, tween 20 for 30 min. Secondary antibodies were then incubated at room temperature for 45 min. After washing the membranes for 30 min, Super Signal West Femto Substrate was added to detect horseradish peroxidase on the membranes. Chemiluminescence was measured and quantified using the Syngene gel imaging system.

Cells were plated in a T25 flask and grown to a 100% confluency. To determine photosensitizer binding, 10 µM of Pd(T4) was incubated for 2 h in a 37 °C incubator with 5% CO₂. Cells were scraped and pelleted at 300× g for 10 min and supernatants were collected for later use. The pellets were washed and resuspended with PBS followed by sonication for 20 s at 30 watts using the F60 Sonic Dismembrator (Fisher Scientific, Hampton, NH, USA). After sonication, each sample was centrifuged at 8160× g for 10 min. Pellets were resuspended in PBS and divided into four treatment groups to determine Pd(T4) molecular associations: 1—control; 2—DNase (1 mg/mL) (Sigma-Aldrich); 3—phospholipase C (12.5 U) (Sigma-Aldrich); 4—trypsin (50 µg/mL). After 37 °C overnight incubation, samples were centrifuged at 8160× g for 10 min and absorption spectra (250–650 nm) were determined for supernatants.

Infected mice were euthanized at respective time points and the MUT tissue was excised and placed in pre-weighed tubes containing 5 mL 1x PBS (-CaCl₂, -MgCl₂) and placed on ice until ready for further processing. Tubes + MUT were weighed to determine the weight of the tissue and the tissue was then minced and resuspended in 1x MUT lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 0.1% Triton X-100, pH 8 + 1x HALT protease inhibitor (Thermo Fisher)) at a volume of 1 mL for every 0.5 g tissue. Lysates were then sonicated using a Sonic Dismembrator F60 (Fisher Scientific) probe sonicator on ice at 60% power for 5 cycles, 0.5 min on/off, set at setting 12 which was followed by centrifugation at 10,000 rpm at 4°C for 10 min to pellet insoluble material. Supernatant was collected and used for Nluc or Bradford assays further described below.