TXNIP Links Innate Host Defense Mechanisms to Oxidative Stress and Inflammation in Retinal Muller Glia under Chronic Hyperglycemia: Implications for Diabetic Retinopathy
Figure 8
HG induces an early ER stress and a later hypoxia-like response in rMC1. Messenger RNA for the spliced form of ER stress marker XBP1 (sXBP1) was measured in rMC1 at various time periods (a) 0–24 h and (b) 0–5 days after HG exposure by qRT-PCR There is no significant change in sXBP1 mRNA (, ) when compared with respective controls at time 0. (c) The DNA-binding activity of sXBP1 in rMC1 was further measured by EMSA using nuclear extracts (5 g) and biotin-labeled DNA probes with or without a competitive cold DNA probe as described in Methods. Protein-DNA complexes were detected by streptavin-HPR and ECL. In the presence of competitive cold probe, the sXBP1 reactive band is abolished indicating the specificity of the sXBP1-DNA binding (last two lanes). Probe alone without the nuclear extract was also run as a control (first lane). The sXBP1 activity is increased at 4 h and 1 day in rMC1 by HG and then returns to the control level observed at day 0. (d) The DNA-binding activity of the hypoxic response factor HIF-1 in nuclear extracts was also measured using a biotin-labeled HIF-1 binding consensus DNA probe. HIF-1 activity is not increased up to day 3 in rMC1 by HG but enhances at day 5. Competitive cold probes block the DNA-binding of HIF-1, indicating the specificity of the binding assay. The images are representative of in both (c) and (d).
