FIGURE 4.
Specific binding of HP to Bac-PrP and Bac-PrPSc. HP-agarose was incubated with the non-seeded and non-reacted iPMCA reaction solution (Bac-PrP), the non-PK-digested reaction solution after PMCA performed using ME7 PrPSc as the seed (Bac-PrP + Bac-PrPSc), and the PK-digested reaction solution after PMCA (Bac-PrPSc, PK-resistant core). After centrifugation, WB was performed to analyze the HP-agarose beads and supernatants, and the band intensities of each fraction were quantified using a ChemiImager. The HP-binding rates are shown as the ratio of Bac-PrP and/or Bac-PrPSc in the bead fraction to total Bac-PrP and/or Bac-PrPSc. Free normal HP (NM) or NaDO was added into the mixtures of each iPMCA reaction solution and the HP-agarose beads during the incubation. Each iPMCA reaction solution was incubated with FLAG-beads (FLAG) as a control. Open circles indicate the binding rates from individual experiments; bars, mean of the binding rates of independent experiments; error bars, S.D. * and #, p < 0.01 (versus without any addition and with addition of free NaDO HP, respectively, in HP-agarose beads). ** and ##, p < 0.001 (versus without any addition and with the addition of free NaDO HP, respectively, in HP-agarose beads). NS and #NS, no significant difference between non-addition and the addition of free NaDO HP and between the addition of free normal HP and the addition of NaDO HP, respectively.