Internalization of PrP^C is dependent on Cdc-42, Rho, Dyn 2, Eps15 and clathrin.

Cells transfected with the different GFP-tagged wt or dominant negative isoforms, with His6-tagged Rho-GDI or siRNA against clathrin heavy chain, were analyzed by confocal microscopy and the results were quantified with Image J software by selecting different optical sections (z stacks, see Materials and Methods) in which we measured the intensity of fluorescence (IF)/unit of area. Data are from experiments repeated three times using 4–5 coverslips for each transfection. The amount of internalized Tfr, PrPC and CTxB (in 30 min of internalization) in the cells transfected with the wild-type or dominant negative GFP-tagged isoforms was expressed as a percentage of the amount in untrasfected cells, which we set as 100% (±S.D. are indicated). The percentages of internalized PrPC and Tfr in siRNA cells for CHC were calculated by measuring the ratio between the average of the values of IF/unit of area in siRNA treated cells (see asterisks in the Figure 3A) and the average values of IF/area in untrasfected cells.