Journal of General Virology

Fig. 3. Western blot presenting the conversion of transiently expressed unglycosylated MHM2PrP(N180Q,N196Q) by brain homogenates in N2a cells. N2a cells were transiently transfected with MHM2PrP(N180Q,N196Q) and inoculated with various quantities of a 10 % RML-infected mouse brain homogenate. The five left lanes show lysates without proteinase digestion; the five right lanes show the same lysates after proteinase K digestion. Background for undigested lysates is high due to overexposure of the film to detect relatively weak protease-resistant unglycosylated MHM2PrP(N180Q,N196Q) (*). Therefore, cross-reactivity is visible between the secondary antibody and cell proteins in the undigested samples on the left side of the immunoblot, and a cross-reactive band of proteinase K (PK) appears in the digested lysates on the right. Expression levels of MHM2PrP(N180Q,N196Q) are about the same (determined by a shorter exposure time of the film for undigested lysates). The upper blot shows an immunoblot with MAb 3F4 that recognizes only newly synthesized MHM2PrP(N180Q,N196Q) (*). Addition of 100 µl of 10 % RML-infected brain homogenate was sufficient to convert MHM2PrP(N180Q,N196Q) into MHM2PrP^Sc(N180Q,N196Q). The lower panel shows the same blot stained with polyclonal antibody R073, which recognizes all PrP, and is used to show that equal amounts of inoculum (**) were present. The inoculum cannot be entirely washed out before lysing the cells.

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