Figure 4.

Time course of antibody-mediated PrP^c internalization. As detailed above (Figure 2), the anti-PrPc antibody-mediated stimulation was achieved by incubating live GN11 cells with Mab 3F4 for 30 min, followed by FITC-conjugated secondary antibody, for a total incubation time of either 60 min (A–D), (M), 90 min (E–H), (N), or 150 min (I–L), (O). After fixing and permeabilizing, cells were probed with anti-Cav-1 Pab and Cy5-conjugated anti-rabbit secondary antibody. The distribution of PrPc (A, E, and I) and Cav-1 (B, F, and J) at the different incubation periods, the superposition (C, G, and K) of the green (PrPc) and red (Cav-1) signals, and the corresponding binary maps (D, H, and L) are shown. Note that after 150 min, the major internalization of PrPc is quite independent from Cav-1 movements (I–L). Panels M–O represent western blot (WB) analysis of cell lysates for the presence of PrP^c glycoforms under the different conditions described. On the right margin, standard molecular weights are reported. For further experimental details see Material and Methods. Bar = 7.5 μm.