FIG. 6.
Effects of wortmannin, a DNA-PK inhibitor, on Sp1-dependent expression. (A) Schematic representations of reporters. p6xSp1CAT contains the HIV-1 minimal promoter (nucleotides −43 to +78) with six consensus Sp1 binding sites positioned upstream of TATAA. pNF-κBCAT contains the HIV-1 minimal promoter and two NF-κB sites positioned upstream of TATAA. (B) Bar graph (average of two experiments) of CAT assays performed in the presence of different concentrations of wortmannin. Activity measured at 0 mM wortmannin was set as 100%. The reporter plasmids (1 μg) are as in panel A. Assays with (pCMV-Tat; right) and without (-pCMV-Tat; left) the addition of a Tat expression plasmid (100 ng) are shown. (C) Cells were treated with the indicated amounts of wortmannin at the same time as with [35S]methionine-cysteine and 32Pi overnight labeling. HeLa cell extracts were immunoprecipitated with anti-Sp1 serum and resolved by SDS-PAGE (10% gel). Benchmark (Life Technologies) molecular weight marker positions are indicated in kilodaltons on the left. Equivalent amounts of total protein were immunoprecipitated as quantitated by TCA precipitation and scintillation counting.