Fig. 2. Tyrphostin A23 blocks PrP^C and transferrin endocytosis but has no effect on ganglioside GM1 endocytosis. (A) SH-SY5Y cells stably expressing PrP^C were seeded onto glass coverslips and grown to 50% confluency. Cells were then pre-incubated with antibody 3F4 at a dilution of 1:1000 in PBS for 30 minutes at 4°C, washed three times in PBS and then incubated for 20 minutes at 37°C in OptiMEM in the absence of Cu²⁺, in the presence of 100 µM Cu²⁺ or in the presence of Cu²⁺ and 500 µM tyrphostin A23. Cells were also incubated with Texas-Red-conjugated transferrin at a dilution of 1:1000. (B) Cells were incubated with BODIPY FL C₅-ganglioside GM1 for 20 minutes in the presence of either 500 µM tyrphostin A23 or 1 mM MßCD. Cells were fixed, incubated with Alexa488-conjugated rabbit anti-mouse antibody and viewed using a DeltaVision Optical Restoration Microscopy System. Images are representative of three individual experiments. Bars, 10 µm.