Cdp star chemiluminescent substrate

Cell lysates in 1% sarcosyl were digested with 75 µg/ml PK (Roche, Switzerland) for 1 h at 37 °C and subsequently denatured in an equal volume of 2x sample loading buffer at 110 °C. Deglycosylation was performed with peptide-N-glycosidase F (PNGase F; New England Biolabs, USA) following the manufacturer’s instructions^{46 (link)}. For this 15 µl PK-digested and denatured samples were mixed with 2 µl each of 10x GlycoBuffer2, NP-40 and PNGase F and incubated for 2 h at 37 °C. 20 µl of either type of samples were run on BOLT 4–12% Bis–Tris mini protein gels for SDS-PAGE and electroblotted using the iBlot 2 dry blotting system (Thermo Fisher, USA). Polyvinylidene fluoride membranes were probed with anti-PrP monoclonal antibody ICSM-18 (1:4000; D-Gen, UK) or 3F4 (1:2000; inhouse production) overnight and anti-mouse IgG alkaline phosphatase-linked secondary antibody (1:5000; Dako, USA). Membranes were incubated with CDP-Star chemiluminescent substrate (Thermo Fisher, USA) for alkaline phosphatase chemiluminescence reaction and detected on Amersham Hyperfilm™ ECL films (GE Healthcare, USA). Images were created in Illustrator 2021 (Adobe, USA).

Western blot analysis for the detection of accumulated PrP^Sc was performed as previously described [4 (link),6 (link)] with modifications. Cells were washed once with PBS and harvested in 100 µL of 1% sarcosyl (v/v). In total, 10 µL of the cell lysate was digested with 75 µg/mL proteinase K (PK; Roche) at 37 °C for 1 h and further denatured in 2× sample loading buffer at 110 °C for 10 min. Ten percent hamster brain homogenate (w/v) of a 263K scrapie-infected animal diluted 1:1000 served as a positive control. SDS-PAGE electrophoresis was performed on BOLT 4–12% Bis-Tris mini protein gels, and proteins were blotted onto PVDF membranes using the iBlot 2 dry blotting system (Thermo Fisher). PVDF membranes were probed with anti-PrP monoclonal antibody 3F4 (1:2000; inhouse production) at 4 °C overnight and anti-mouse IgG alkaline phosphatase-linked secondary antibody (1:5000; Dako, Santa Clara, CA, USA). Protein bands were detected with CDP-Star chemiluminescent substrate (Thermo Fisher) for alkaline phosphatase chemiluminescence reaction on Amersham Hyperfilm^TM ECL films (GE Healthcare, Chicago, IL, USA), which were exposed for 40 min.