Fig. 2
Prion protein misfolding activity detected by sPMCA in PBMC of recipient sheep transfused with monocytes or T lymphocytes from scrapie-affected sheep. PBMC (1x107 cells) were added to mNBH and subjected to six PMCA rounds of 48 cycles of sonication and incubation. Samples were diluted 1:3 into fresh mNBH between rounds. Pre- and post-PMCA samples underwent proteinase K digestion (200 μg/ml for 90 min at 37 °C) prior to western blot analysis with prion mAb P4. Representative western blots are shown. a PBMC prepared from positive control scrapie sheep (4124) pre- and post-sPMCA (lanes 1 and 2, respectively). PBMC prepared from monocyte recipient sheep (lane 4 = 4546, lane 5 = 4593, lane 6 = 4594, lane 7 = 4595, lane 9 = 4597) and an uninoculated control sheep (lane 8 = 4596). A scrapie naïve sheep (lane 3 = 4545) and mNBH (lane = 10) served as negative controls for sPMCA. b PBMC from positive control scrapie sheep (4124) is shown pre- and post-sPMCA (lanes 1 and 2, respectively). PBMC prepared from T lymphocyte recipient sheep (lane 4 = 4539, lane 5 = 4588, lane 6 = 4589, lane 7 = 4590) and uninoculated control sheep (lane 8 = 4591, lane 9 = 4597); and mNBH (negative sPMCA control, lane = 3). Molecular mass markers (in kDa) are indicated on the left of the blots