EMSA using the nuclear extracts of IL-11– and heparin-treated calvaria cells.

Calvaria cells were either left untreated (lane 2) or stimulated with 25 μg/mL heparin (lane 3), 20 ng/mL IL-11 (lanes 4, 6, 7, 8), or a combination of heparin and IL-11 (25 μg/mL and 20 ng/mL, respectively; lane 5) for 10 minutes at 37°C. Nuclear extracts were then prepared and EMSA was performed as described in “Materials and methods.” The EMSA was performed with probe alone (no nuclear extracts; lane 1), and competition was performed with excess (100 ×) unlabeled homologous oligonucleotide (lane 6) or mutated oligonucleotide differing from the SIE by 3 nucleic acids (lane 7), whereas supershifts were performed with anti-STAT3 Ab (lane 8). The arrow indicates STAT3 homodimerization and its association with radiolabeled SIE.