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Fig. 3. Effects of the antioxidant N-propylgallate (nPG; 100 µM, preincubation for 30 minutes before treatment) on the stability of HIF-1 protein during reoxygenation in the salmonid cell lines RTG-2 and CHSE-214. In both cell lines, hypoxia treatment (1% O₂ for 2 hours) caused stabilization of HIF-1 protein. Consequent reoxygenation (15 minutes at 5, 10 and 21% O₂) caused a reduction in the level of HIF-1 protein in the absence of the antioxidant, whereas reoxygenation in the presence of nPG prevented the reduction of HIF-1 protein level completely. (A) A representative gel showing the intensities of HIF-1 protein bands at the different conditions. HIF-1 protein was detected using immunoprecipitation techniques with HIF-1 antibody produced against rainbow trout protein. (B) Quantification of HIF-1 protein band intensities in normal oxygen conditions before the experiment (21% oxygen), in hypoxia (1% oxygen), during reoxygenation (5, 10 and 21% oxygen) and during reoxygenation in the presence of nPG (N5%, N10% and N21%). The intensity of HIF-1 protein band in normal oxygen at the onset of the experiments was taken to be 100%. ANOVA, carried out using SPSS statistical software, indicated that oxygen had a significant (P<0.01) effect on the HIF-1 protein band intensity during reoxygenation in the absence but not in the presence of nPG. Values represent mean±s.e.m. (n=4). Similar results were obtained using NAC (10 mM) as the antioxidant.

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