Fig. 5. Ad.IBAS inhibits IB protein expression and elevates baseline levels of NFB in the nucleus. HeLa cells were infected at an moi of 1000 particles/cell with Ad.IBAS or Ad.GFP. Cytoplasmic and nuclear extracts were prepared from cells collected at 24, 48 and 72 hours after infection. (A) 5 µg of cytoplasmic protein was evaluated by western blotting using anti-IB antibody, anti-IBß antibody or anti-actin antibody. Ad.IBAS infection led to a reduction in IB protein expression at all time points (lane 2-4) and an increase in IBß expression compared with Ad.GFP-infected controls (lane 1). Changes in IB expression were referenced to actin protein levels, which did not change. (B) 5 µg of nuclear protein was evaluated by EMSA to determine NFB DNA-binding activity. The induced p65-p50 heterodimer of NFB, identified by supershift assays, is marked by an arrow. Results in A and B are derived from the same experimental samples, and experimental conditions are marked above each lane. (C) 5 µg of nuclear protein from 72 hour post-infection time points with Ad.GFP (same sample as from lane 2 Panel B) and Ad.IBAS (same sample as from lane 5 Panel B) were evaluated by EMSA supershift to determine the subunit composition of the NFB DNA binding complex. Antibodies used for supershift were against p50, p52, C-Rel, Rel B and p65. The active DNA-binding complex is identified as p65-p50 heterodimers, and supershifted bands (in lanes 2, 6, 8 and 12) are marked by an asterisk to the left of the gel.