Internalization of PrPC 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�C5 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.