Fig. 2 | Communications Biology

Fig. 2

From: Early stage prion assembly involves two subpopulations with different quaternary structures and a secondary templating pathway

Fig. 2The alternative text for this image may have been generated using AI.

Seed concentration- and time-dependent dynamic evolution of the PMCA-generated PrPSc assemblies. a, b SV profiles of mb-PMCA products seeded with serial ten-fold dilutions from 127S-infected brain homogenates, as indicated. Thirty minutes after the last sonication, the amplified products were solubilized and SV-fractionated. The mean relative levels of PK-resistant PrPSc per fraction were obtained from the immunoblot analysis of n = 4 independent fractionations of PMCA reactions (a, representative dot-blot shown). Variation in the P1 and P2 peak maximum (mean ± SEM values) as a function of the logarithm of the seed dilution factor (b). c PK-resistant PrPSc sedimentograms from the PMCA products generated with 127S prions (10−5 dilution) and further incubated at 37 °C during the indicated quiescent phase (t), i.e., without sonication. At each time point, the collected products were frozen prior SV analysis. All collected samples were then thawed, fractionated in parallel by SV and analysed by immunoblot (c, n = 3 independent experiments, representative dot-blot shown). d PK-resistant PrPSc isopycnic sedimentograms from PMCA products generated with 127S prions (10−5 dilution) and immediately fractionated at the end of the PMCA reaction (blue line and symbol) or after a 24-h-quiecent incubation at 37 °C (red line and symbol). At each time point, the collected samples were frozen. All collected samples were then thawed, fractionated in parallel by sedimentation at the equilibrium20 and analysed by immunoblot (the mean ± SEM levels of PK-resistant PrPSc per fraction were obtained from the immunoblot analysis of n = 3 independent fractionations of PMCA reactions). As control, the density profile of PK-resistant PrPSc assemblies from the brain of terminally sick tg338 mice infected with 127S prions (solubilization at 37 °C to mimic the PMCA conditions) is shown (gray line and symbol). e Evolution of the percentage of P1 and P2 peak surface areas as a function of the quiescent phase post-PMCA reaction (c). f PK-resistant PrPSc sedimentograms from the PMCA products generated with 139 A and vCJD prion seeds (10−5 dilution) and further incubated for a quiescent period of 48 h at 37 °C (mean ± SEM values from n = 3 independent experiments)

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