Fig. 2
From: Two alternative pathways for generating transmissible prion disease de novo
Generating rPrP fibril-induced atypical PrPres in dgPMCAb. a Analysis of PK-resistant materials produced in 6th round of serial PMCAb or dgPMCAb reactions seeded with four preparations of rPrP fibrils or non-seeded reactions by Western blot. F2M or F0.5 are fibrils generated in 2Â M or 0.5Â M GdnHCl, respectively, as previously described [11]. FBSA or FNBH are fibrils annealed in BSA or NBH as previously described [10, 29]. b Analysis of PK-resistant materials produced in 6th round of serial dgPMCAb reactions seeded with four independent preparations of F0.5 fibrils or non-seeded reactions by Western blot. c Establishing a limiting dilution for atypical PrPres in S05 brain material. S05 brain material was subjected to 10-fold serial dilutions for up to 1013-fold and used for seeding serial dgPMCAb, using a procedure previously described [34]. Western blot analysis of 18th serial dgPMCA rounds demonstrates that 109-fold diluted S05 brain material was the last dilution that contained atypical PrPres material. d To confirm that preparation of brain-derived atypical PrPres subjected to 18th dgPMCA rounds lacks PrPSc, serial PMCAb reactions were seeded with the products of 18th dgPMCAb round or 109-fold diluted S05 brain material, then subjected to six serial PMCAb rounds and analyzed by Western blot. PK-resistant bands at 23, 16 and 13Â kDa represent di-, mono- and unglycosylated atypical PrPres, respectively. Western blots in panels a-c were stained with SAF-84 antibody and in panel d with 3F4 antibody