E64 inhibits C3 generation and PrPres truncation but not PrPres biosynthesis. (A) E64 treatment lowers steady-state PrPres levels after a 3-d treatment of 22L(230TC) cells with the indicated concentration of inhibitor. PrPres was detected by immunoblot. The bands in each lane correspond to the three glycoforms of PrP. The graph shows the average intensity (± SEM) of the PrPres bands as a percentage of the untreated control (n = 3). (B) Influence of E64 on metabolism of FlAsH-PrP(230TC). The fluorescent gels and the graphs show the results of FlAsH-pulse-chase analysis of 22L(230TC) cells incubated in the absence or presence of E64 (80 μg/ml). The graphs show average band intensity ± SEM (n = 4). The double asterisk denotes a statistically significant difference in C3 levels at 6 h between E64-treated and untreated samples (two-tailed unpaired t test, p = 0.0089). The arrows indicate full-length FlAsH-PrP(230TC), the arrowhead indicates the 21-kDa truncated PrPres band, and the asterisk indicates the C3 fragment. Lysate, kinetics of FlAsH-PrP(230TC) in cell lysate samples (lanes 1–11). PTA, kinetics of FlAsH-PrP(230TC) in PTA-precipitated samples (lanes 12–24). Ch+, samples digested with chymotrypsin (lanes 23–24). Full, full-length FlAsH-PrP(230TC). C3, C3 fragment. 21 kDa, 21-kDa truncated PrPres band.