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. 2017 Aug 29;7:9584. doi: 10.1038/s41598-017-09489-3

Figure 7.

Figure 7

In vitro propagation of human GSS-associated misfolded rec-PrPs by brain-PMCA. (A) The GSS P102L-129M misfolded rec-PrP was selected as the seed to be subjected to 3 serial rounds of brain-PMCA using brain homogenates from Tg340 mice or TgNN6h mice as substrates. A brain homogenate from a GSS P102L-129M affected patient was used as control seed. The study was performed in quadruplicates and unseeded samples for each brain-based substrate were used as negative controls. Neither the GSS P102L-129M misfolded rec-PrP nor the GSS brain-derived seed were able to propagate the misfolding to the Tg340-derived human PrPC. However, the same misfolded rec-PrP was propagated efficiently (see the four positive duplicates) with the unglycosylated human protein (TgNN6h-derived PrPC). A PK (at 85 µg/ml) resistant fragment of approximately 20 kDa, in contrast to the approximately 17 kDa of the seed, suggests the existence of a GPI anchor as a standard characteristic of prions propagated in brain-derived substrates. Unseeded samples from both substrates remained negative until at least 5 PMCA rounds suggesting the absence of spontaneous misfolding or cross-contamination. The GSS patient control was run in a separate gel and the image shown, divided from the other gel by a vertical grey line, was cropped adjusting the size in accordance with the molecular weight markers. (B) The four GSS-associated misfolded rec-PrPs were selected as seeds to be subjected in quadruplicate to 10 serial rounds of brain-PMCA (R1-R10) using brain homogenates from TgNN6h mice as substrate. A representative experiment for each round is shown after PK (85 µg/ml) digestion of the samples. Unseeded tubes containing only TgNN6h based substrate were used as control for cross-contamination. Three GSS-associated misfolded rec-PrPs, including both polymorphic variants of P102L and A117V 129M, required only 3 brain-PMCA rounds for full adaptation to the unglycosylated substrate. In contrast, the A117V-129V misfolded rec-PrP needed 10 serial PMCA rounds to be propagated. 3F4 monoclonal antibody (1:10,000) was used. Mw: Molecular weight.