Fig. 4. Expression of glycosylated FLAG-tagged human and bovine PrP in BHK cells transfected with recombinant SFV RNAs. (A) Constructs of human PrP containing a FLAG-tag insertion either between amino acids 227 and 228 close to the GPI-anchor adhesion site or behind the N-terminal signal peptide between amino acids 22 and 23 and a bovine PrP construct with the FLAG-tag inserted between amino acids 239 and 240. (B) Expression and deglycosylation of wild-type and FLAG-tagged human PrP. (C) Expression and deglycosylation of wild-type and FLAG-tagged bovine PrP. Total cell extracts from SFV-transfected BHK cells were incubated overnight with N-glycosidase F at 37°C and analyzed by western blotting. For detection, mAb anti-PrP 3B5 was used. FLAG-tagged prion proteins were glycosylated in the same manner as wild-type PrP (B, lane 2 and 3 and C, lane 2). After treatment with N-glycosidase F, the glycosylated forms are clearly reduced, accompanied by an increase in the non-glycosylated forms (B, lane 4, 5 and 6; C, lane 3 and 4). The shift in the molecular weight of non-glycosylated FLAG-tagged PrPs is due to the additional eight amino acids of the FLAG-tag. (D) Effects of endoglycosidase H on FLAG-tagged human and bovine PrP. Total cell extracts were incubated for 3 hours with Endo H at 37°C and analyzed by western blotting using mAb 3B5. Endo H sensitivity is shown by an increase of the non-glycosylated PrP form (D, lanes 2, 4 and 6).