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Edward Chaum, Jinggang Yin, Weihong Huo, John C. Lang; High Throughput Protein Assays of Gene Expression Following Oxidative Stress in Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4440.
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We have previously shown that oxidative stress (OS) can quantitatively regulate AP-1 gene expression in RPE cells. In the present study we explore novel strategies for quantifying the protective effects of antioxidants using transcription factor (TF) and antioxidant protein biomarkers in a high throughput, cell-based assay.
ARPE-19 cells were seeded in 96-well plates and grown to confluence in defined media to stabilize gene expression. Cells were treated with 100µM or 350µM H2O2 for up to 6 hours and AP-1 family, EGR1/2, HO-1, and β-actin proteins were detected using IRDye 800CW-labled antibody. Fixed cells were permeabilized, labeled, and scanned at 700nm and 800nm wavelengths using an Odyssey imager to quantify biomarker and control protein levels.
We quantified the expression of the AP-1 transcription factors, EGR1/2, HO-1, and a β-actin control. Following 100µM and 350µM H2O2 OS, AP-1 protein FosB increased 13% and 23%, cFos increased 15% and 48%, ATF3 increased 18% and 35%, and JunB increased 28% and 27% respectively, compared with the control wells. These data correlated well with our previously published Western results showing a dose-dependent increase in mRNA and protein levels in response to OS.
The high throughout cell-based Western assays may be a useful method to quantify OS-induced gene expression in RPE cells. The 96-well plate format permits us to sample the expression of target proteins under different incubation and stress conditions, and to evaluate temporal changes in protein expression over time in a high throughput manner.
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