Fig. 5.
In vitro propagation ability of ovine and cervine recombinant misfolded seeds on substrates containing ovine rec-PrP with substitutions that define the cervine rigid loop (S173N T177N) and with other mule deer substitutions in combination with the ones defining the rigid loop. Western blots showing PK-resistant misfolded rec-PrP after serial dilutions from 10−2 to 10−8 of OvDawson, OvBSE, and MuCWD rec-PrPres and a single 48 h rec-PMCA round on substrates containing ovine rec-PrP with substitutions (N177T), (S173N N177T), (S98T N177T), (I208M N177T), (S98T S173N N177T), and (I208M S173N N177T) complemented with chicken brain homogenate. The negative control samples (0) were unseeded ovine ARQ rec-PrP substrate. NA indicates the no-amplification sample with the inocula diluted 10−1 and not subjected to sonication. The rec-PMCA products were digested with PK (25 μg/ml) for 1 h at 42 °C, analyzed by Western blot, and probed with 9A2 antibody (diluted 1:4000). Recombinant ovine or cervine PrPs (rec-PrP) and tg338 brain homogenate (Tg338) were used as controls. PK, Proteinase K; MW, molecular weight. Below the panel of Western blots is a representative experiment of three replicates, and maximum dilutions for each strain are plotted as an average of three independent experiments, including standard deviations. The Western blots and results corresponding to N177T substitution are also presented in the previous figure due to its relevant blocking effect in order to facilitate the interpretation of results in both figures