Figure 4.
PrPSc strings on the surface of living cells resist extraction by PIPLC and colocalize with GM1 and caveolin. (A) Live ScGT1 and ScGT1-PPS cooled to 8°C were stained with 8B4 followed by RRX secondary Fabs. (c and d) Cells were treated with PIPLC (1 U/ml; for 1 h at 8°C) before staining. (B) Live ScGT1 were labeled at 8°C with Fab EST123 followed by secondary Fab (monovalent, to prevent Ab-induced PrP patching). (C) GT1 and ScGT1 cells chilled to 8°C were treated or not with PIPLC and incubated with 8B4, and cell-bound mAb was then quantified in a WB developed with secondary Abs coupled to HRP. In infected cells, PIPLC reduced the amount of cell surface–bound 8B4 by ∼50% (lanes 2 and 4). (D, a–d) On the surface of live ScGT1 cells, 8B4-decorated strings (a and c, red) largely overlapped with CTxB (b, arrows) but not with TfR (d, circles and arrowheads). (e and f) Colocalization of 8B4 strings (red) with caveolin 1 (green) in fixed and permeabilized SMB cells treated with GdnSCN. (E) SMB contain caveolin-1. Positive control: C6 glioma.