FIG. 3.

Kinetics of MCP-1 and IL-8 inhibition by Ad E3. U373 cells (10⁶) were grown in α-MEM medium supplemented with 10% FBS or depleted of serum for 14 h prior to infection. Cells were subsequently infected with 4,000 particles of virus per cell; 10 ng of TNF-α per ml was added 4 h prior to harvesting each sample at the time points indicated. (A) RNase protection assay. The first lane is a negative control (no virus or TNF). The time of harvest of the mock- or AdCMVE3 (E1 and E3 positive)-infected cells and the presence or absence of serum are indicated for each lane. (B) Western blot. U373 cells were infected with Ad7001 and AdCMVE3 and some samples were treated with TNF-α 4 h prior to harvesting at 12 or 16 hpi for protein and analysis as described in Materials and Methods. The infecting adenovirus and the presence of serum or TNF stimulation is indicated for each lane. The Ad E3-gp19K band, detected by rabbit polyclonal antibodies, is labeled