FIGURE 5.

PrP^res from supernatants is not fully soluble. A, left, PK-digested lysates from infected Rov cells were centrifuged in a TLA-110 rotor at 20,000 or 100,000 × g for 1 h. Various portions of the corresponding supernatants (Sup^20K or Sup^100K, respectively) were analyzed for PrP^res by Western blotting. Right, undigested lysates from uninfected Rov cells were analyzed for normal PrP before (−) or after (+) 100,000 × g centrifugation. Please note that these ultracentrifugation conditions correspond to 100,000 × g or 500,000 × g with the SW32 rotor used previously (see “Experimental Procedures”). B, soluble fraction of a PK-digested cell lysate was ultracentrifuged for 1 h at 200,000 × g in a TLA-110 rotor either directly (−) or through a 10% (w/v) sucrose cushion (+). The pellets were analyzed for PrP^res by Western blotting.