PTX does not inhibit preactivated (in whole cells) NF-κB binding with its motif
Jurkat cells were grown under the standard tissue culture conditions in the presence of PMA (20 ng/ml, 12 hr). Nuclear extracts were prepared and EMSA was conducted with 10 µg of nuclear proteins and 20,000 cpm of 32P-labeled oligonucleotide carrying the NF-κB motif, under conditions previously described (3). The figure shows the binding activity in nuclear extracts of PMA-treated cells in the presence of the indicated concentrations of PTX added to the cell-free binding reaction. As previously reported from this laboratory (3,42), nuclear extract isolated from cells treated with PMA and PTX at these same concentrations showed inhibition of NF-κB binding with its motif. The binding activity was determined by densitometric scanning and integration of the specific autoradiographic signal as described above.