Nuclear NF-κB activity determined by EMSA. Nuclear proteins of JPX-9 cells treated with (B; CdCl₂ treatment for 6 hours) or without CdCl₂ (A) in the presence or absence of NF-κB inhibitors, was incubated with radiolabeled oligonucleotide containing NF-κB binding site, and nuclear NF-κB activity was examined by EMSA as described in the text. (A) Nuclear NF-κB activity in CdCl₂-untreated JPX-9 cells. Lane A; positive control (MT-2 cells). Lane B; CdCl₂-untreated JPX-9 cells without NF-κB inhibitors. Lane C; CdCl₂-untreated JPX-9 cells, and then cultured in 50 μmol/L of PDTC for 3 hours. Lane D; CdCl₂-untreated JPX-9 cells, and then cultured in 50 mmol/L of NAC for 3 hours. Lane E; CdCl₂-untreated JPX-9 cells, and then cultured in 10 μmol/L of LLL-CHO for 3 hours. Note that basal nuclear NF-κB activity in CdCl2-untreated JPX-9 cells was clearly suppressed by 3 kinds of NF-κB inhibitors. (B) Nuclear NF-κB activity in CdCl₂-treated JPX-9 cells. Lane A; control (CdCl₂-untreated JPX-9 cells without NF-κB inhibitors). Lane B; CdCl₂-treated JPX-9 cells (without NF-κB inhibitors). Lane C; CdCl₂-treated JPX-9 cells, and then cultured in 50 μmol/L of PDTC for 3 hours. Lane D; CdCl₂-treated JPX-9 cells, and then cultured in 50 mmol/L of NAC for 3 hours. Lane E; CdCl₂-treated JPX-9 cells, and then cultured in 10 μmol/L of LLL-CHO for 3 hours. Lane F; CdCl₂-treated JPX-9 cells, and then cultured for 3 hours without NF-κB inhibitors. Note that nuclear NF-κB in JPX-9 cells was clearly augmented by CdCl₂ treatment, and its activity is still high in the presence of 3 kinds of NF-κB inhibitors. (C) Nuclear NF-κB activity of Jurkat cells was not augmented by incubation of the cells with CdCl₂. Lane A; Jurkat cells without CdCl₂ treatment. Lane B; Jurkat cells treated with CdCl₂ for 6 hours. Results (Fig 8A through C) are representative of 5 experiments.