Figure 3
PK-titration assay. Supernatant (S1) from each of the three sCJDVV subtypes was digested with PK concentrations of 0.6 to 160 U/ml, as indicated, and probed with 3F4 (A,B), 12B2 and To-2 (C,D) or 12B2 (E). (A) PK1/2 (index denoting amount of PK in Units/ml required to digest half of resPrPD) for sCJDVV1–2 (45 ± 3 U/ml) was about intermediate between that of T1 in -VV1 (27 ± 4 U/ml) and that of T2 in -VV2 (106 ± 13 U/ml) (PK1/2 VV1 vs. VV2, P < 0.0006; PK1/2 VV1–2 vs. VV1, P < 0.03; PK1/2 VV1–2 vs. VV2, P < 0.02). (B) Representative WB of resPrPD obtained at the indicated PK concentrations from each of the three sCJDVV subtypes and used to generate the curves of (A,C) S1 from the three sCJDVV subtypes probed with the T1- and T2-specific Abs 12B2 and To-2. Hydrolysis profiles of T1 and T2 from sCJDVV1–2 mimic those of T2 from -VV2 and T1 from -VV1, respectively. PK1/2 in T1 from -VV1 and -VV1–2 are 18 ± 5 U/ml and 8.3 ± 1.5 U/ml, respectively (P > 0.05); PK1/2 in T2 from -VV2 and VV1–2 are 130 ± 15 U/ml and 142 ± 26 U/ml, respectively (P > 0.05). The peculiar double exponential equation profile observed with To-2 likely relates to the specific and efficient detection by this Ab of PrP residue 97 only when this residue is exposed as N-terminus; this condition requires a distinct initial digestion phase20. (D) Representative WB of T1 (12B2; green dye) and T2 (To-2; red dye) obtained at the different PK-concentrations in the three sCJDVV subsets. (E) PK1/2 determination of T121 and T120 in the absence and presence of T2. A significant reduction in PK-resistance occurs in T121 in the presence of T2 (PK1/2 31.5 to 10 U/ml) while T120 maintains the comparable PK1/2 (8.2 and 6.7 U/ml).
