Effects of ischemia or hypothermia on DNA binding activity. (A) Electrophoretic mobility shift assay (EMSA) analysis of metal regulatory transcription factor 1 (MTF-1) or metal response elements d (MREd) binding activity was performed using nuclear extract proteins from the normal control, after 4 hours of oxygen glucose deprivation (OGD), and 3 hours after reperfusion at 37°C or 33°C. MTF1-DNA complexes were formed by incubating the biotin-labeled MREd probe with different nuclear extract proteins. The mixture was then separated in a non-denaturing polyacrylamide gel. The shifted bands (arrow) corresponding to the MTF-1/DNA complexes were visualized relative to the unbound double-stranded DNA. Competition experiment was performed as follows: a. OGD-stimulated sample with labeled MTF-1 probe. b. OGD-stimulated sample with cold and labeled MTF1 probe. c. Dexametasone-stimulated sample with labeled MTF-1 probe. DNA binding activity increased after OGD with both normothermia and hypothermia. DNA binding activity is expressed as mean ± S.E.M. of four experiments. *P <0.05 compared to the control. (B) EMSA analysis of MREa binding activity. A competitive experiment was performed as follows: a. Positive control with labeled control probe. b. Positive control with labeled probe and cold probe. c. OGD-stimulated sample with labeled MRE probe. d. OGD-stimulated sample with cold and labeled MRE probe. e. Dexametasone-stimulated sample with labeled MRE probe. MRE binding activity was increased by both ischemia and hypothermia. Binding activity is expressed as the mean ± S.E.M. of four experiments *P <0.05 compared to the control; #P <0.05 compared to normothermia. (C) STAT3 binding activity was measured using a STAT3 transcription factor assay kit. Hypothermia increased the binding of STAT3. Binding activity is expressed as the mean ± S.E.M. of four different experiments. *P <0.05 compared to the control; #P <0.05 compared to normothermia.