Pvdf membrane

Cells were lysed and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of protein were separated on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Germany). Membranes were blocked and then incubated with a primary antibody against DLL3 (Abclonal, China, A18108), CD63 (Abclonal, China, A19023), CD81 (Abclonal, China, A4863), TSG101 (Abclonal, China, A1692), Calnexin (Abclonal, China, A4846). Following primary incubation, membranes were treated with HRP-conjugated secondary antibody (Abclonal, China, AS014). Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Meilunbio, China) and quantified with image analysis software.

Cell lysates from U251 and U87 lines were obtained through brief sonication within a modified RIPA solution (Biosharp, China), enhanced with inhibitors for proteinases and phosphatases. Protein levels within these lysates were quantified employing a BCA protein assay kit (Beyotime Biotechnology, China), adhering to the protocol provided by the manufacturer. Following this, protein separation was achieved through 10% SDS-PAGE, with the proteins then being transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% non-fat milk at room temperature for 2 hours and was then incubated overnight at 4°C with primary antibodies (anti-γ-H2A.X HL1299, GeneTex; anti-GAPDH 60004-1-Ig, anti-Erk1/2 11257-1-AP, anti-pErk1/2 28733-1-AP and anti- TSPAN13, 18974-1-AP, ProteinTech, China; anti-JAK2 #3230, anti-pJAK2 #3774, anti-STAT3 #12640, anti-pSTAT3 #9145, CST). After three washes with 1 × TBST, the membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (SA0001-1 or SA0001-2, ProteinTech, China) for 2 hours. The immunoreactions were visualized using ECL reagent, and the immunoblots were developed to detect the target proteins.

All fawns were tested for CWD by tonsil biopsy; all tested negative. The deer were housed individually in concrete rooms that had not been previously used for TSE studies. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the School of Veterinary Medicine Animal Care and Use Committee at the University of Wisconsin (Permit Number: V910). Bucal swabs were obtained from each deer and the PRNP genes amplified and sequenced as described previously [4] (link).
The deer were dosed daily, for five consecutive days, with 20 ml 10% (w/v) pooled brain homogenate. The pooled brain homogenate was prepared from obex brain samples obtained from two CWD-positive Wisconsin hunter-harvested deer. These deer were both wt/wt with respect to PRNP genotype and were histologically scored as stage 4 positive in the obex [4] (link). The inoculum was prepared in phosphate-buffered saline and was mixed with two cups deer pellet feed and fed to the deer. Additional feed was withheld from the deer for the five days of oral infection to ensure the complete consumption of inoculum.
Brains from each animal were homogenized (20% w/v) in cold PBS (DNase I 250 µg/ml) in a blender and then passed through different size needles. Aliquots of brain homogenate from each genotype were digested with proteinase K (50 µg/ml) for 30 minutes at 37°C, reactions were stopped by boiling in SDS sample buffer at 95°C for 10 minutes. The samples were resolved by western blot, using 12% NuPAGE Bis-tris gels (Invitrogen, CA) and PVDF membrane (Millipore). Blocking was performed in 5% milk in 0.1% TBS-T for 1 h at room temperature. Incubation with primary antibody, 8G8 1∶5000 (Cayman Chemical), was performed overnight at 4°C and HRP secondary antibody was used at 1∶10,000. Images were captured in a Typhoon system after ECL substrate addition (Pierce).
Obex immunohistochemistry was performed as described [4] (link). Briefly, samples were fixed in 10% neutral buffered formalin, dehydrated and embedded in paraffin. Tissue sections (5 µm thick) were cut and placed on positively charged slides. Slides were deparaffinized and antigen retrieval was performed by hydrated autoclaving in retrieval buffer. The tissue sections were exposed to anti-PrP mAb 6H4 (Prionics, Switzerland). Primary antibody was detected using a biotinylated secondary anti-mouse antibody, followed by horseradish peroxidase–streptavidin conjugate, chromagen substrate and hematoxylin counterstain.