FIG. 1.

Detection of an abundant secreted protein in supernatants of γHV68 infected fibroblasts. A murine 3T12 fibroblast cell line was either mock infected or infected at an MOI of 5 with γHV68 virus (WUMS strain) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% fetal calf serum (FCS). After 1 h, the monolayer was washed with phosphate-buffered saline, fresh DMEM containing 1% FCS was added back to the monolayer, and the infection was allowed to proceed for 23 h. The culture supernatant was passed through a 0.2-μm-pore-size filter to remove any cellular debris and was concentrated at 4°C by using an Amicon (Beverly, Mass.) Centriprep-10 concentrator (from 15 ml to 600 μl). In addition, the concentrated supernatants were centrifuged at 150,000 × g for 3 h to remove any remaining aggregated material and residual free virus present in the supernatant samples. The concentrated supernatant samples, as well as the high-speed supernatants and the pellets obtained from the 150,000 × g spin, were analyzed on SDS–12% polyacrylamide gels. (A) SDS-PAGE analysis and staining with Coomassie brilliant blue R250. Note the prominent ca. 45-kDa band in the γHV68-infected precentrifugation and γHV68 ultracentrifugation supernatant lanes and the absence of this band in both the mock-infected supernatant and the virus-infected high-speed pellet. (B) Immunoblot analysis of fractions shown in panel A, probed with a rabbit polyclonal anti-γHV68 antiserum (25) (1:2,000 dilution), followed by a 1:5,000 dilution of horseradish peroxidase-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch, West Grove, Pa.), and visualized with a chemiluminescence detection kit (Amersham, Arlington Heights, Ill.). No reactive species were detected when normal rabbit serum was used as a control primary antibody (data not shown). Molecular sizes in kilodaltons are given in the center. The arrow indicates the migration of the virus-specific band. The prominent band above the virus-specific band is serum albumin.