Figure 5. Upregulation of ubiquitin following exposure to oxidative stress

mRNA for three ubiquitin genes was determined by northern blot (A) and real time RT-PCR (B). A: Northern blot analysis of endothelial cells exposed to 40 mU/ml glucose oxidase for 4 h shows three different-sized transcripts representing Ubiquitin A52 (0.7 kb), Ubiquitin A+B (0.8-1.5 kb), and Ubiquitin C (2.5 kb). UbA52 corresponds to a ubiquitin-ribosomal fusion protein, whereas UbB and UbC correspond to doublet and linear repeats of polyubiquitin chains, respectively. The content of mRNA for ubiquitin is normalized against the signal for actin. In endothelial cells exposed to glucose oxidase, mRNA of ubiquitin A and ubiquitin A+B increase as compared to the controls. Ubiquitin C mRNA is not significantly different in cells exposed to oxidative stress (p<0.05). Each bar represents the mean±SE of five independent experiments. B: mRNA levels of the ubiquitin genes were detected by quantitative RT-PCR. In endothelial cells exposed to glucose oxidase, mRNA of ubiquitin B increases compared to the controls. Ubiquitin A52 and ubiquitin C mRNA are not significantly different in cells exposed to oxidative stress (p<0.05). Each bar represents the mean±SE of four independent experiments.