Journal of General Virology

Fig. 4. Western blot presenting the conversion of transiently expressed unglycosylated MHM2 PrP(N180Q,N196Q) by different strains of mouse prions in N2a cells. The seven left lanes show lysates without proteinase digestion; the seven right lanes show the same lysates after proteinase K digestion. Background of 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 as determined by a shorter exposure time of the film for undigested lysates. N2a cells were transiently transfected with different MHM2 PrP constructs as indicated and inoculated with an identical quantity (150 µl) of different mouse brain homogenates. The upper blot shows an immunoblot with MAb 3F4 that recognizes only newly synthesized MHM2PrP constructs. Inoculation of MHM2PrP(N180Q,N196Q) expressing N2a cells with 10 % brain homogenates of different mouse prion strains, RML, Me7 and 301V, converted MHM2PrP(N180Q,N196Q) equally well to MHM2PrP^Sc(N180Q,N196Q) (*). Neither the negative control (no inoculation) nor the control inoculation with normal CD-1 brain homogenate converted PrP(N180Q,N196Q). Also, inoculation of MHM2PrP expressing N2a cells did not result in a significant conversion; a faint band with the electrophoretic mobility of unglycosylated PrP^Sc can be seen after proteinase K digestion, indicating that the unglycosylated population of PrP glycoforms is most readily converted into PrP^Sc. As for ScN2a cells (see Fig. 2), addition of another mutation Q218K to MHM2PrP(N180Q,N196Q) blocked conversion. 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 cell lysis.

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