FIGURE 4.

Membrane attachment, but not lipid raft localization, is necessary for ΔCR PrP toxicity. A, shown is surface immunofluorescence staining of PrP on HEK cells stably expressing ΔCR, ΔCR/GPI(−), ΔCR/M6PR, or WT/M6PR. Scale bar = 50 μm. B, a Western blot detects PrP secreted into the growth medium by cells expressing ΔCR and ΔCR/GPI(−) PrP. C, an Opti-Prep gradient to separate DRMs is shown. ΔCR PrP is present at the 0/30% Opti-Prep interface (lanes 4–5), as is the DRM marker flotillin-1. ΔCR/M6PR PrP is present in the fractions containing 40% Opti-Prep (lanes 12–14). Sample from the total cell lysate (L) is shown in lane 1. D, whole-cell patch clamp recordings at −80 mV were made from cells expressing the indicated constructs. E, quantitation of the currents recorded in panel D plotted as the percentage of total time the cells exhibited an inward current ≥450 pA (mean ± S.E., n = 5 cells). F, cell death induced by G418 treatment (400 μg/ml) measured by MTT reduction (mean ± S.E., n ≥ 10 wells from three independent experiments). Asterisks (*) indicate values that are significantly greater than those for WT PrP (p < 0.05, one-tailed Student's t test).