FIG. 7.
Immunoblots of soluble or insoluble cell lysates from vJB11-infected cells. Approximately 2 × 106 CV1 cells were mock infected or were infected with pUL6 null virus, vJB11, or HSV-1(F). At 18 hours postinfection, cells were washed with cold PBS and solubilized in either (i) 800 μl of SDS sample buffer (whole-cell lysates) (A) or in 400 μl of RIPA buffer (B to E). After extensive clarification of the RIPA-fractionated material, proteins in the supernatant (soluble) (B and D) or pellet (insoluble) (C and E) were denatured in SDS, separated on an SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with pUL6 (A, B, and C) or scaffold-specific antibodies (D and E). Bound immunoglobulins were revealed by reaction with appropriate secondary antibodies and enhanced chemiluminescence. (F) The chemiluminescent intensities corresponding to the position of the pUL6-containing band in each lane of panels A, B, and C were determined using an LAS-3000 Mini Fujifilm imaging system and are reported as a percentage of that obtained in panel A, lane 4. (G) Similar to panel F, but intensities of ICP35 signals were normalized to that in panel D, lane 4.