Figure 1.

EMSA analysis showing changes in DNA binding activity of NF-κB in the nuclear extracts of hepatocytes (A) and IHECs (B) stimulated via CD40 or TNF-α (positive control). Aliquots of nuclear extracts (20 μg of protein) from cultured and stimulated primary human hepatocytes and IHECs were subjected to EMSA analysis with ³²P-labeled oligonucleotide probe consensus sequence to NF-κB. In hepatocytes, NF-κB activation was detected at 2 h but was largely undetectable at 24 h (A), whereas NF-κB activation was sustained >24 h in the IHECs (B). (A) Hepatocyte NF-κB: lane 1, negative control (labeled probe alone); lane 2, 2-h TNF-α stimulation; lane 3, unstimulated control; lane 4, 2-h CD40 stimulation; lane 5, supershift by using anti-RelA mAb; lane 6, cold competition with 100× excess of unlabeled probe; lane 7, 24-h CD40 stimulation. (B) IHEC NF-κB: lane 1, negative control (labeled probe alone); lane 2, 2-h unstimulated control; lane 3, 2 h after TNF-α stimulation; lane 4, 2-h CD40 stimulation; lane 5, 24-h CD40 stimulation; lane 6, cold competition with 100× excess of unlabeled probe.