Fig. 4

a Effect of neem leaf extract on the binding affinity of NF-κB assessed by an EMSA. K562 cells were pretreated during 2 h with different concentrations of neem leaf extract (80–320 μg/ml), followed by TNF-α addition (at 20 ng/ml). After additional 6 h of incubation, the cells were extracted by a method described by Müller and coworkers. [38]. The DNA binding affinity is determined by an incubation of the nuclear cell extract (10 μg) with a labeled oligonucleotide probe containing the NF-κB binding site C-κB. b A quantification of one representative experiment is shown. c In case of a super-shift/immunodepletion experiment, the nuclear extract was first incubated with the antibody in the mixture for 15 min, before the labeled probe was added, followed by an incubation period of 30 min. Positive control was a cell solution treated with TNF-α only. Negative control was a cell solution with neither TNF-α nor natural compound. The data shown here were representative for three independent experiments with similar results