FIG. 3.

Immediate-early gene expression is sufficient for loss of IκBα. (A) Replicate cultures of CV-1 cells were mock infected or infected with KOS at an MOI of 5 under the conditions indicated and described in Materials and Methods. Lysates were prepared at 8 hpi, and the accumulations of indicated proteins were determined following fractionation on 12% polyacrylamide gels. The lysates were sequentially probed for IκBα, VP16, and α-tubulin or for ICP0 and ICP8. HSV/Cx rev, infection of cells in the presence of cycloheximide followed by removal of cycloheximide and addition of actinomycin D (ActD) at 5 hpi; HSV+PAA, infection of cells in the presence of PAA. (B) Replicate cultures of CV-1 cells were mock infected or infected with wt KOS or the ICP4 mutant vi13 (MOI = 5). Lysates were prepared at 8 hpi, fractionated on 12% polyacrylamide gels, and sequentially probed for IκBα and α-tubulin or for ICP4 plus ICP0 and ICP8. NS, nonspecific. (C) Relative amounts of IκBα from three independent experiments were determined using Image J and averaged, as described in Materials and Methods.