rhHMGB1 induces nuclear translocation and DNA binding of transcription factors NF-κB and SP-1.
Nuclear extracts were prepared from HMEC-1 cells after 4-hour incubation with control medium alone (lanes 1-2), TNFα 10 ng/mL (lanes 3-4), and rhHMGB1 100 ng/mL (lanes 5-8). The nuclear extracts were incubated with 32P-labeled NF-κβ or32P-labeled Sp1 consensus oligonucleotides and resolved by EMSA. Unlabeled oligonucleotide (100 ×; either NF-κβ or Sp1) was added to lanes 2, 4, and 6. Nuclear extracts from the rhHMGB1-treated HMEC-1 cells showed increased binding of the NF-κB (a) and Sp1 (b) oligonucleotides compared with untreated cells. Antibodies against NF-κB subunits p50 and p65 were added to lanes 7 and 8, respectively. The arrow (c) in lane 8 shows a supershift for the NF-κB p65 subunit. Representative results of 4 independent experiments for both NF-κB or Sp1 are shown.
