Fig. 6

SNOC inhibits DNA binding activity of zinc finger transcription factor Sp1. Gel shift experiments were performed with Sp1-specific ³²P-labeled oligonucleotide probes (sequence indicated below) and recombinant Sp1 (A) or nuclear extracts from EL4-6.1 cells (B, C). (A) Recombinant Sp1 was incubated in the absence (Ctrl) or presence of 0.5 mM, 1 mM, or 2 mM of SNOC or 2 mM of SNOC_−NO for 30 min at room temperature. Subsequent electrophoretic mobility shift assays revealed that SNOC, in contrast to SNOC_−NO, decreased the Sp1 binding activity in a concentration-dependent manner. (B) Nuclear extracts of IL-1β-treated EL4-6.1 lymphoma cells were treated without (Ctrl) or with 1 or 2 mM of SNOC or with 2 mM of SNOC_−NO for 30 min at room temperature. As with the recombinant Sp1, SNOC, in contrast to SNOC_−NO, inhibited the DNA binding activity of nuclear Sp1. (C) EL4-6.1 lymphoma cells were incubated for 3 hr without (Ctrl) or with 1000 U/ml IL-1β in the absence or presence of 0.5 mM or 1 mM of SNOC, 1 mM of SNOC_−NO, or 0.5 mM of H₂O₂. Subsequently, nuclear extracts were prepared. SNOC (0.5 and 1 mM), in contrast to SNOC_−NO or 0.5 mM of H₂O₂, greatly reduced the binding of nuclear Sp1 to its consensus oligonucleotide. Representative experiments are shown.