Figure 5. Characterization of PrP^C in gradient fractions obtained from treated CGCs.

Panel A Cells, after incubation with different gangliosides (GM3, GM1 or GD1a) and correspondent radiolabelled gangliosides ([³H]GM3, [³H]GM1 or [³H]GD1a), at a final concentration of 2×10⁻⁶ M at 37°C for 4 h (Standard treatment, St), were treated with 1% Triton X-100-containing buffer for 30 min on ice. The cellular lysate was submitted to discontinuous sucrose density gradient centrifugation. One-milliliter fractions were withdrawn from the gradient, submitted to 15% SDS-PAGE (20 µg protein/lane), transferred to nitrocellulose membranes and immunoblotted with 6H4 or SAF32 antibodies against PrP^C followed by ECL detection. Representative blots from three independent experiments are shown. g: glycosylated PrP^C; u: unglycosylated PrP^C. GM3 = GM3-treated CGCs; GM1 = GM1-treated CGCs; GD1a = GD1a-treated CGCs. Panel B-E: immunofluorescence analysis of PrP^C in CGCs with 6H4Ab (B and C) and SAF32Ab (D and E) in the presence (C and E) or in the absence (B and D) of GM1. Note that after ganglioside treatment, PrP^C recognized by 6H4Ab appears generally less clusterized being more widespread and less concentrated around cell bodies and proximal dendrites (C, green), while PrP^C distribution detected by SAF32Ab does not differ from that of control cells. Insets show double staining of PrP^C and CTB. Arrows mark the position of CTB. Scale bar: 10 µm; insets: 20 µm.