EMSA of STAT6 binding activity. The DNA binding activity of nuclear extracts from HepG2 cells cultured in the presence or absence of IL-1β and/or IL-4 for 30 minutes were examined using a 32P-labeled oligonucleotide from the sIL-1Ra promoter region containing the recently characterized STAT6 binding element (SBE1). Competition studies were performed with nuclear extracts from HepG2 cells stimulated with IL-4 (see Materials and Methods). Lane 1, unstimulated; lane 2, IL-1β; lane 3, IL-4; lane 4, IL-1β and IL-4; lane 5, competition with the cold probe; lane 6, competition with the cold probe mutated in the potential STAT6 binding site; lane 7, competition with the cold oligonucleotide containing another potential STAT6 binding site within the sIL-1Ra promoter (SBE2); lane 8, competition with the cold oligonucleotide containing the STAT6 consensus element; lane 9, preincubation of nuclear extracts with antibodies against STAT6 before the addition of the32P-labeled SBE1 probe; lane 10, preincubation of nuclear extracts with control IgG. Lanes 11 and 12 contain the SBE1 probe and the antibodies against STAT6 or control IgG, respectively, without nuclear extracts. The dark arrow shows the STAT6-DNA complex. The open arrow represents the supershifted complex. NS = nonspecific protein-DNA binding. These results are representative of three different experiments.
