FIG. 7.
Activation of NF-κB in TGEV-infected ST cells. (A) EMSAs were performed with a total extract containing 4 μg of protein by using a 32P-labeled oligonucleotide with a consensus κB sequence (see Materials and Methods). The arrow indicates the NF-κB-specific complex. (B) The specificity of the retarded complexes was assessed by preincubating an extract with an excess of an unlabeled (cold) NF-κB or an unrelated SP1 oligonucleotide (oligo) probe, as indicated. The slower-migrating band indicated by the arrow disappeared completely when an unlabeled NF-κB competitor was included, indicating that this complex is specific for the NF-κB probe. The faster-migrating band indicated by the circle was partially competed by unlabeled NF-κB or an unrelated SP1 probe, indicating that it is not specific for the NF-κB probe. (C) Analysis of the subunit composition of the major complex induced by TGEV infection. Specific antibodies (Ab) to human p50 and murine p65 were added to the total binding mixture as described in Materials and Methods. The arrow indicates the NF-κB complex. Supershifted complexes are indicated by the arrowhead. (D) Inhibition of NF-κB binding activity by NF-κB decoy experiments. Double-stranded phosphorothioate oligonucleotides used as TFDs contain three copies of the consensus κB sequence (26). TFDs were added to the cell medium to a concentration of 3 μM 24 h before TGEV infection. At 4 and 9 h p.i., total cell lysates were prepared and subjected to EMSAs as described for panel A. The arrow points to the major NF-κB retarded complex induced by TGEV infection. Note that the faster-migrating band indicated by the circle was poorly affected by the TFDs, confirming that it is not specific for the NF-κB probe.