Figure 2

Hcy treatment elicited NF-κB activation in Neuro2a cells. A) Detection by EMSA of NF-κB DNA binding activity in nuclear extracts of untreated or Hcy-treated Neuro2a cells. After a 4 h incubation with 250 μM Hcy, in the presence or absence of either IRFI 016 (80 μM) or the NF-κB specific inhibitor SN50 (50 μg/ml), NF-κB activation was evaluated by incubation of nuclear proteins (6 μg) with a 40-mer biotinylated oligonucleotide probe containing the NF-κB consensus sequence. B) Densitometric analysis of NF-κB activated complexes. The amount of NF-κB activated complexes was significantly increased by Hcy treatment. Notably, IRFI 016 as well as SN50 strongly reduced Hcy-increased NF-κB levels. Results shown were from three separate experiments. *p < 0.05 significant differences in comparison with untreated cells, and ^§§p < 0.01 significant differences in comparison with Hcy-treated cells. C) Supershift analysis of NF-κB activated complexes. A supershift assay, carried out by incubating nuclear proteins with specific monoclonal antibodies against p50 and p65 NF-κB subunits, demonstrated that NF-κB activated complexes were p50/p65 heterodimers.