Fig. 4
MR100 shows oligomer-inducing activity in brain homogenates from prion-infected rodents. a MR100 oligomer-inducing activity was tested using freshly homogenized rodent brain tissues infected with the 22 L (mice) or the 263 K prion strain (hamsters). Fifty μL of 10 % mouse or hamster brain homogenates were diluted in 300 μL PBS/2 % Sarkosyl, incubated with 1.5 mM MR100 (corresponding to 150 μL of 5 mM MR100) at room temperature for 1 h or with 150 μL of DMSO as control, and then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins). PK digestion was stopped by addition of a cocktail of protease inhibitors (Complete), before analysis of rPrPSc by western blotting with the SAF mix according to the previously described protocol [16]. CTR: untreated 22 L- or 263 K-infected brain homogenates (negative control); DM: 22 L- or 263 K-infected brain homogenates incubated with DMSO. b Comparison of P30 and MR100 oligomer-inducing activity on the 22 L prion strain, before and after proteinase K digestion. Fifty μL of 10 % 22 L-infected brain homogenates were diluted in 350 μL PBS/2 % Sarkosyl, incubated with 1 mM MR100 or P30 (corresponding to 100 μL of 5 mM MR100 or P30), at room temperature for 1 h. Then, aliquots of 30 μL were taken before addition of proteinase K, to perform western blot (PK-) probed with SAF mix, but also with anti-β-actin antibodies as loading controls. The rest of the sample was then digested with 20 μg/ml PK at a ratio of 1:50 (PK/proteins) (PK+). The reaction was stopped by addition of the protease inhibitor cocktail, before analysis of rPrPSc by western blotting with the SAF mix as in A. DM: 22 L-infected brain homogenates incubated with 100 μL DMSO. Molecular masses (20–50 kDa) are indicated on the left side of the panels