Figure 3.

(A) Mock- and 0.3 mM FeCl₂-exposed cell lysates were fractionated by differential centrifugation and immunoblotted with 3F4. As expected, the majority of PrP^C from mock-treated lysates partitions in the detergent-soluble S₁I and S₂I fractions (lanes 1 and 3), with minimal amounts in the P₁I and P₂I fractions (lanes 2 and 4). In contrast, significant amounts of PrP^C from FeCl₂-treated lysates are detected in the pellet fractions P₁I and P₂I (lanes 6 and 8). A minor 20-kDa fragment is observed in the P₁I fraction of treated cells (lane 6). (B) Reprobing with anti-ferritin shows up-regulation of ferritin in FeCl₂-treated lysates (lanes 1–8), 40% of which partitions in the P₂I fraction (lane 8). (C) FeCl₂ treatment has no detectable effect on the expression or solubility of β-actin. (D) Treatment of lysates prepared from mock- and FeCl₂-exposed cells with 7.5 and 15 μg/ml PK results in the complete digestion of PrP^C in mock-treated controls (lanes 1–3), whereas treated lysates show a 20-kDa PK-resistant fragment (lanes 4–6). (E) Reprobing with anti-ferritin shows up-regulation of ferritin in FeCl₂-exposed cells (lanes 1–6) and the generation of a 14-kDa fragment by PK (lanes 5 and 6, arrow).