FIG 3.

Steady-state NID1 protein levels declined rapidly in lysates and supernatants. (A) HFFs were mock- or virus-infected at a multiplicity of infection (MOI) of 5 and harvested for Western blot analysis at the indicated times pi. Lamin B served as a loading control in all blots. (B) Supernatants were harvested from mock- or virus-infected HFFs at 72 hpi. An additional aliquot from mock-infected cells was placed in a fresh dish and sampled every 24 h for an additional 96 h. (C) Equivalent amounts of supernatant aliquots (100 μl) were harvested from a single plate (with 8 ml of medium) of either mock- or virus-infected HFFs at the indicated times pi and analyzed by Western blotting. (D) HFFs were mock- or virus-infected at an MOI of 5. At 3.5 hpi, virus was removed, and medium containing 0.5 μM MG132 (or dimethyl sulfoxide [DMSO] vehicle) was added to inhibit proteasomal activity. Cells were harvested for Western blot analysis at the indicated times pi.