FIG. 3.
Protease digestion of MHM2(Δ23–88,Δ141–221). (A) Lanes A1 through A3, N2a cells transfected with pSPOX[MHM2(Δ23–88,Δ141–221)]; lanes B1 through B3, mock-transfected N2a cells; lanes C1 through C3, 0.4 μg of myristylated, synthetic MHM2(Δ23–88,Δ141–221) peptide per ml mixed with untransfected N2a cell lysate (0.5 mg of total protein per ml). Lanes A1, B1, and C1, untreated whole-cell lysates; lanes A2, B2, and C2, pellet fractions of lysates digested with 7 μg of proteinase K per ml for 30 min at 37°C; lanes A3, B3, and C3, pellet fractions of lysates digested with 20 μg of proteinase K per ml for 1 h at 37°C. SDS-PAGE was performed on 16% Tricine gels (Novex). Western blotting was performed with 3F4 MAb at 1:5,000 dilution. After processing, lanes 1 and 2 were exposed for 1 min and lane 3 was exposed for 15 min to Hypermax film (Amersham Life Sciences). Units are apparent molecular sizes based on migration of protein standards in kilodaltons. (B) Proteinase K digestion of brain homogenates from Tg mice. Lane 1, 60-day-old, uninoculated Tg(PrP106)Prnp0/0 mouse; lane 2, 65-day-old, scrapie-infected Tg(PrP106)Prnp0/0 mouse; lane 3, 48-day-old, spontaneously ataxic Tg(PrP61)Prnp0/0 mouse. Minus symbols denote undigested control sample, and plus symbols designate the pellet fraction of sample subjected to digestion with 20 μg of proteinase K per ml for 1 h at 37°C. Units are apparent molecular sizes based on migration of protein standards in kilodaltons.