Estudo Funcional de Mutantes da Proteína Prion Celular Associados à Doenças por Prion

We initially tested whether in these cells STI1 would also in- duce endocytosis. Immunofluorescence analysis of cell-surface PrP3F4 indicates that STI1, but not STI1<l230 –245, decreased cell- surface immunolabeling for PrP C (Fig. 2C). In contrast, in N-PrP3F4-expressing cells, treatment with STI1 did not change cell-surface immunolabeling (Fig. 2 D). To complement these ex- periments and quantify the effect of STI1, we used biotinylation of cell-surface PrP C (Fig. 2 E). In these experiments, we measured the amount of PrP3F4 remaining at the cell surface after the cells were treated with STI1 (Ribeiro et al., 2005; Lee et al., 2007). In control experiments, cells exposed to Cu 2+ showed a decrease in cell-surface PrP C (63 ± 10% of decrease; mean ± SEM for seven experiments). Treatment of cells with STI1 for 5 min caused se- questration of PrP3F4 with consequent decrease in cell-surface PrP C (Fig. 2 E) (34 ± 7%; mean ± SEM for six experiments). In contrast, N-PrP3F4 cells treated with STI1 showed no sign of internalization (Fig. 2 E) (2 ± 2%; mean ± SEM of four experi- ments). Thus, the combination of live cell, flow cytometry, im- munofluorescence, and biotinylation experiments showed that STI1 was able to induce PrP C endocytosis in two distinct neuro- nal cell lines.

Incubation of PrP-null CF-10 cells with STI1 induced neither


PrP C-null, Prnp0/0 primary hippocampal neurons (B), CF-10 and CF-10 PrP3F4 cells (D); or

SN56 cells…