Influence of calphostin C and H88 on NF-κB binding and HIV-1 LTR-driven reporter gene expression in PMA- and TNF-α-treated 293-27-2 cells
293-27-2 cells were grown to semiconfluency under standard tissue culture conditions. Cells were incubated in the presence of calphostin C (under light) or H88 at the indicated concentrations for 30 min. This was followed by treatment of the cells with PMA (20 ng/ml) or TNF-α (10 ng/ml) at 37°C for an additional 3.5 hr in 5% CO2 and 95% air. An equivalent volume of DMSO was added to control plates (untreated cells). The cells were harvested and cytoplasmic and nuclear extracts were prepared as described previously (3). NF-κB binding activity in 5 µg of nuclear extract were determined by EMSA and µ-galactosidase activity in 10 µg of cytoplasmic extract of control and treated cells were determined as described previously (3). Binding activity of NF-κB was determined by densitometric scanning and integration of the specific autoradiographic signal as described in Fig. 3. Percent inhibition was calculated by considering NF-κB binding and β-galactosidase activity in the absence of drug as 0% inhibition.