FIG. 6.

Purification of virions and Western blotting for U14 and p53. (A) PCR assay to detect viral DNA in fractions from the first step of purification by sucrose gradient centrifugation. Fractions of 500 μl each were collected from the bottom, diluted 1:100 in Tris-EDTA buffer, and subjected to PCR using the primer pair 6U14-1B and 6U14-2H amplifying part of the U14 ORF. (B) The virions purified from the sucrose gradient centrifugation were subjected to Western blotting to confirm the purity. Fraction 9 was resolved by SDS-PAGE with whole-cell lysates of mock (m)- and HST (i)-infected Molt-3 cells at 72 hpi. Molecular weights of protein markers are shown at the left side of each gel. (C) Virions purified by the second step of purification by discontinuous CsCl gradient centrifugation were subjected to Western blotting with antibodies against U14, p53, gL, and IE1 and to PCR using the same primer set used above. (D) Fractions 8 and 20 were analyzed by silver staining. Molecular weight markers (lanes M) are indicated at the left of the gel. IB, immunoblot.