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. 2019 Apr;189(4):797–812. doi: 10.1016/j.ajpath.2018.12.016

Figure 2.

Figure 2

Time course effect of lipopolysaccharide (LPS) on Caco-2 NF-κB pathway (p65 and p52) activation. A: LPS at the concentration of 300 pg/mL caused significant increase in p65 protein expression in Caco-2 cells on day 3 to 3.5. The expression of p52 and p100 did not change with LPS treatment. β-Actin was used as an internal control for protein loading. B: Relative densitometry analysis for p65 protein levels. C: Confocal immunofluorescence of Caco-2 cells treated with 300 pg/mL LPS (3 to 3.5 days) indicated p65 (red) translocation to the nucleus (blue) (arrowheads). D: P52 (green) did not change after LPS treatment (arowheads). E: LPS caused degradation of inhibitory κ B (IκB)-α expression (3 to 3.5 days), as assessed by Western blot analysis. β-Actin was used as an internal control for protein loading. F: Densitometry analysis of LPS treatment showed significant decrease in IκB-α level on day 3 to 3.5 compared with control untreated cells. G and H: LPS-treated Caco-2 cells (3 to 3.5 days) analyzed by flow cytometry for phospho-IκB-α and phospho-p65, respectively, showed increased expression compared with control untreated cells. Gray indicates isotope control (Cont); blue, control untreated; red, LPS treated day 3. [Mean fluorescence intensity (MFI): pIκB-α, 175 ± 12.12, versus 292 ± 25.63; pp65, 3307 ± 154.7, versus 4329.3 ± 169.4]. Data are expressed as means ± SEM. n = 3 independent experiments. ∗∗∗P < 0.001 versus control; †††P < 0.01 versus LPS (day 4); ‡‡‡P < 0.01 versus LPS (day 3.5). Scale bars = 5 μm (C and D).