Fig. 3. PrPwt and T182A mutant have different intracellular distributions in FRT cells. (A) FRT cells expressing PrPwt and T182A were grown on transwell filters for 4 days and fixed with 2% paraformaldehyde. Saponin permeabilized (P) or non-permeabilized (NP) samples were treated with the -PrP antibody (PRI308) and the secondary TRITC-conjugated antibody. The samples were then examined in a Zeiss laser scanning confocal microscope (LSCM 510). Z (upper panels) and horizontal (lower panels) sections are shown. Bar, 10 µm. (B) FRT cells expressing PrPwt (left panels) and T182A (right panels) were grown on coverslips in semiconfluent conditions, fixed and permeabilized with 0.075% saponin. Then they were incubated with the -PrP mAb (PRI308) and with primary polyclonal antibodies against different markers of intracellular compartments, e.g. calnexin (CNX), giantin and early endosomal antigen 1 (EEA1) and then treated with -mouse and -rabbit secondary antibody conjugated with TRITC or FITC. Lysotracker was used to label lysosomes for 1 hour in vivo before fixation and confocal imaging. Bar,10 µm. Note that in the semiconfluent cells used for these experiments, which are optimal to visualize intracellular compartments, no plasma membrane signal was found for PrPwt (B). By contrast in polarized filter grown cultures the majority of the protein appears to be at the cell surface (A).