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Figure 4: Acid induces nuclear factor-kappa B in esophageal epithelial cells. (a) Effect of acid on nuclear factor-kappa B binding activity. OE33 cells were incubated in media of different pH values ranging from pH 7.4 to pH 4.0. The pH of the medium was adjusted by adding 0.1 M HCl. OE33 cells were exposed to low pH for 1 h and nuclear extracts were prepared and assayed for nuclear factor-kappa B binding activity by electrophoretic mobility shift assay. (b) Effects of acid on IκB-α protein levels. OE33 cells were exposed to increasingly acidic conditions as indicated and total cell extracts were prepared and analyzed by Western blotting for IκB-α proteins using specific antisera. (c) Time-course of nuclear factor-kappa B induction by acid. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. Nuclear extracts were prepared and gel shift assays for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (d) Time effect of acid exposure on IκB-α protein levels. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. The cells were collected for the preparation of total cell extracts. Western blots for IκB-α were performed on total cell extracts. A representative gel of three independent experiments with similar results is shown. (e) Supershift and competition assays were carried out on nuclear extracts from OE33 cells stimulated at pH 6.8. The binding reaction was performed after 30 min incubation with or without 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, c-Rel, lane 4 and 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5. Each experiment was repeated 3 times with similar results and a representative gel is shown

Figure 4: Acid induces nuclear factor-kappa B in esophageal epithelial cells. (a) Effect of acid on nuclear factor-kappa B binding activity. OE33 cells were incubated in media of different pH values ranging from pH 7.4 to pH 4.0. The pH of the medium was adjusted by adding 0.1 M HCl. OE33 cells were exposed to low pH for 1 h and nuclear extracts were prepared and assayed for nuclear factor-kappa B binding activity by electrophoretic mobility shift assay. (b) Effects of acid on IκB-α protein levels. OE33 cells were exposed to increasingly acidic conditions as indicated and total cell extracts were prepared and analyzed by Western blotting for IκB-α proteins using specific antisera. (c) Time-course of nuclear factor-kappa B induction by acid. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. Nuclear extracts were prepared and gel shift assays for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (d) Time effect of acid exposure on IκB-α protein levels. OE33 cells were incubated in media of pH 6.8 for different periods of time as shown. The cells were collected for the preparation of total cell extracts. Western blots for IκB-α were performed on total cell extracts. A representative gel of three independent experiments with similar results is shown. (e) Supershift and competition assays were carried out on nuclear extracts from OE33 cells stimulated at pH 6.8. The binding reaction was performed after 30 min incubation with or without 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, c-Rel, lane 4 and 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5. Each experiment was repeated 3 times with similar results and a representative gel is shown