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Figure 2: Dose- and time-dependence of nuclear factor-kappa B activation by deoxycholic acid. (a) Dose-response of nuclear factor-kappa B activation by deoxycholic acid. OE33 cells were treated with different concentrations of deoxycholic acid (300 μM) as indicated. Nuclear extracts were prepared and gel shift assay for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (b) Effect of deoxycholic acid doses on IκB-α protein levels. OE33 cells were treated with deoxycholic acid at the indicated concentrations, and Western blotting for IκB-α was performed. (c) Time-course of nuclear factor-kappa B induction by deoxycholic acid. OE33 cells were treated with deoxycholic acid (300 μM) 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 deoxycholic acid exposure on IκB-α protein levels. OE33 cells were treated with deoxycholic acid for different periods of time, and Western blotting for IκB-α was performed. (e) Supershift assay and competition assays. Supershift assay was performed using 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, and c-Rel, lane 4. Competition assay for nuclear factor-kappa B was also performed using 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5

Figure 2: Dose- and time-dependence of nuclear factor-kappa B activation by deoxycholic acid. (a) Dose-response of nuclear factor-kappa B activation by deoxycholic acid. OE33 cells were treated with different concentrations of deoxycholic acid (300 μM) as indicated. Nuclear extracts were prepared and gel shift assay for nuclear factor-kappa B binding activity were performed using a radiolabeled nuclear factor-kappa B probe. (b) Effect of deoxycholic acid doses on IκB-α protein levels. OE33 cells were treated with deoxycholic acid at the indicated concentrations, and Western blotting for IκB-α was performed. (c) Time-course of nuclear factor-kappa B induction by deoxycholic acid. OE33 cells were treated with deoxycholic acid (300 μM) 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 deoxycholic acid exposure on IκB-α protein levels. OE33 cells were treated with deoxycholic acid for different periods of time, and Western blotting for IκB-α was performed. (e) Supershift assay and competition assays. Supershift assay was performed using 0.5 μl of rabbit antisera to p50, lane 2, p65, lane 3, and c-Rel, lane 4. Competition assay for nuclear factor-kappa B was also performed using 100-fold molar excess of unlabeled nuclear factor-kappa B, lane 5