Advertisement

< Back to Article

Unraveling the key to the resistance of canids to prion diseases

Fig 5

Two cell/brain-PMCA methodologies to study the effect of the residue 158 in mouse PrP.

A. Schematic representation of the cell/brain-PMCA propagation study. Brain-derived PrPC (black filled circles) is mixed with cell-derived 3F4-tagged PrPC (grey filled circles) and seeded with PrPSc (RML or 22L, black filled squares). The resulting 3F4-tagged PrPSc (grey filled squares) is specifically detected by the 3F4 antibody. B. A 1:40 dilution of RML or 22L were used as seeds for a PMCA based on mouse brain homogenate mixed with cellular 3F4-tagged substrates containing mouse PrP N158, D158, E158 or without PrP (PRNP0/0; KO-PrP). Non-PMCA amplified samples and samples subjected to one 24 h single round of PMCA were digested with PK (20 μg/ml) and analyzed by Western blot using monoclonal antibody 3F4 (1:10,000). Control: undigested human brain homogenate. C. Schematic representation of the cell/brain-PMCA inhibition studies. Brain derived PrPC (black filled circles) is mixed with cell derived PrPC (black filled circles) and seeded with PrPSc (RML, black filled squares). Total resulting PrPSc is detected by antibody D18. D. 1:5,000 or 1:10,000 dilutions of RML were used as seeds for a PMCA based on mouse brain homogenate mixed with cellular substrates containing mouse PrP N158, D158, E158 or without PrP KO-PrP. Samples subjected to one 24 h single round of PMCA were digested with PK (20 μg/ml) and detected by the monoclonal antibody D18 (1:10,000). Both procedures showed a significant inhibitory effect of the D158 and E158 substitutions over the in vitro propagation of RML/22L mouse prion strains. Control: undigested mouse brain homogenate. Mw: Molecular weight.

Fig 5

doi: https://doi.org/10.1371/journal.ppat.1006716.g005