Figure 5
From: The non-octarepeat copper binding site of the prion protein is a key regulator of prion conversion
Biochemical properties and cellular localization of H96Y mutant in N2a cells.
(a) The H96Y mutant displays mild PK-resistance when expressed in N2a cells regularly passaged every 7 days up to passage (P) 8. Cell lysates were treated with 2 or 5 μg/mL of PK. Two exposures of the same blot are shown (Faint: 30 sec exposure; Dark: 6 min exposure). PrPs were detected by anti-PrP 3F4 antibody. β-actin is used as internal control. Arrows (▼) indicate positions on the gels where the blots have been cropped. Full-length blots are presented in the Supplementary Figure S5a–b. (b) Schematic representation of the seeding experiment in N2a cells. (c) PTA-extracted PrPSc from N2a cells transfected with 3F4-H96Y MoPrP were inoculated into N2a cells and regularly passaged every 7 days up to P8. The PrPres detection was assessed by PK digestion (5 μg/mL) through passages. Full-length blots are presented in the Supplementary Figure S5c–d. (d) ThS-positive H96Y MoPrP aggregates detected in N2a cells. The cells were stained for PrP expression (red) and ThS (green). Untransfected N2a and ScN2a cells (mock) were used as controls. Scale bar: 12 μm.
