Figure 4

TLR2 ligands induced PI3K-Akt-dependent regulation of intestinal barrier function in IPEC-J2 cells. IPEC-J2 cells were stimulated with PI3K inhibitor, LY294002 (10 μg/mL) for 1 h prior to the treatment with PCSK and LTA-BS for 24 h. A Total Akt (serine 473), p-Akt, p70S6K, Bcl-2, FAK, and β-actin were examined using Western blot assay. B TJ proteins, claudin-3 and ZO-1 compared to β-actin control were examined for their expression from whole-cell lysates by Western blot assay. The representative figure from four similar results is shown. C The expression of claudin-3 and ZO-1 in the cells was visualized using confocal microscopy after staining with anti-claudin-3 or -ZO-1 antibody conjugated with Alexa fluor 488-FITC (green) and nuclei (DAPI; blue). The representative figure from four similar results is shown. D The cells were pre-treated with TLR2 neutralizing antibody (20 μg/mL) for 1 h before TLR2 stimulation in order to neutralize TLR2. DON (2 μg/mL) was treated for 48 h, and the expression of claudin-3, occludin, and ZO-1 was visualized using confocal microscopy. The representative figure from four similar results is shown. Scale bar = 50 μm.