Inhibition of the binding activity of activated NF-κB by PTX with cytoplasmic extracts from Jurkat and 293-27-2 cells
Conditions for the PKC- and PKA-catalyzed activation of NF-κB in a cell-free system were as described in the legend to Fig. 1. Two micrograms of fractionated cytoplasmic protein from Jurkat (upper panels) and 293-27-2 cells (lower panels) was used in the presence of indicated units of purified PKA (A) or purified PKC (B) per 5 µl of reaction mixture. (C) show percent inhibition of PKC (0.2 U/5 µl reaction of mixture)- or PKA (2 U/5 µl reaction mixture)-catalyzed production of active NF-κB in a cell-free system in the presence of increasing concentrations of PTX. The level of active NF-κB in the presence of increasing concentrations of pure enzyme was quantitated by densitometric scanning and integration of the specific autoradiographic signal on an LKB 2222-020 Ultroscan XL Laser Densitometer (LKB Instruments, Houston, TX, U.S.A.). Binding activity (numerals on the y axis) represents the relative intensities of the specific DNA-protein interaction with the 32P-oligonucleotide-containing NF-κB motif (described in Materials and Methods) and the trans-activator.