FIGURE 7.

The effect of chemical antagonist of S1PR2 on S1P- and TCA-induced activation of IKKα/β-NF-κB pathway. HuCCT1 cells were cultured in serum-free medium overnight and pre-treated with JTE-013 (10 μm) for 1 h, and then treated with S1P (100 nm) or TCA (100 μm) for 4 h. A and B, total protein was isolated, and protein levels of p-IKKα/β, total IKKα/β, p-NF-κB p65, and total NF-κB p65 were determined by Western blot analysis. The relative densities of p-IKKα/β/total IKKα/β and p-NF-κB p65/total NF-κBp65 were determined. C and D, cytosol and nuclear proteins were isolated as described under “Experimental Procedures.” Protein levels of NF-κB p65, lamin B, and actin were determined by Western blot analysis. Lamin B and actin were used as a loading control for nuclear and cytosol protein, respectively. A and C, representative images of the immunoblots for p-IKKα/β, total IKKα/β, p-NF-κB p65, and total NF-κB p65 are shown. B, relative protein levels of p-IKKα/β/total IKKα/β and p-NF-κB p65/total NF-κB p65. D, relative protein levels of nuclear NF-κB p65/lamin B and cytosol NF-κB p65/actin. Values represent the mean ± S.E. of three independent experiments. Statistical significance relative to the vehicle control: *, p < 0.05; **, p < 0.01; ***, p < 0.001; relative to S1P or TCA treatment group: #, p < 0.05; ##, p < 0.01; ###, p < 0.001.