Figure 4. Hydrogen sulfide counteracted ischemia/reperfusion-induced DNA-binding activity of nuclear factor-kappaB . Effect of hydrogen sulfide (H₂S) on the DNA-binding activity of nuclear factor-kappaB (NF-κB) in retinal tissue 24 h after unilateral ischemia. Control eyes with and without H₂S preconditioning did not reveal any DNA binding of NF-κB (lanes 1, 3). Compared to control, ischemia/reperfusion (I/R) injury increased the DNA binding of NF-κB significantly (lane 1 versus 2; sixfold induction versus control, p<0.001). H₂S inhalation before I/R injury counteracted the ischemia-induced DNA binding of NF-κB completely (lane 4 versus 2, 5.8-fold reduction versus I/R injury, ***p<0.001). Specific supershift analysis revealed that p50 is the main part of the transactive NF-κB domain (lane 6), while the unspecific antibody (c-fos [AP-1]) did not lead to a supershift. The oligo’s sensitivity was demonstrated by competition experiments with unlabeled NF-κB (lane 7) and unlabeled AP-1 (lane 8). The histogram represents the densitometric ratio of NF-κB compared with the control group. Data are presented as mean±SD of five experiments.