Effect of gliadin peptide 10-mer on p31–43 activity and entrance in CACO-2 cells.

<p>(<b>A</b>) CACO-2/TC7 cells, either unstimulated or stimulated with p31–43 (50 µg/ml), p10-mer (50 µg/ml) + p31–43 (50 µg/ml), were analyzed by Western blot for ERK phosphorylation and NF-kB activation. (<b>i</b>) Phosphorylated levels of ERK were analyzed in whole cell extracts by Western blot with anti-phospho-ERK1/2 antibodies; for control, the blotted membranes were stripped and reprobed with anti-ERK1/2 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>ii</b>) NF-kB activation was analyzed in whole cell extracts by Western blot with anti-phospho-NF-kB p65 Ser antibodies; for control, the blotted membranes were stripped and reprobed with anti-NF-kB p65 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software, peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments. (<b>B</b>) CACO-2/TC7 cells, either unstimulated or stimulated with biotinylated p31–43 (50 µg/ml), p10-mer (50 µg/ml) + biotinylated p31–43 (50 µg/ml), were immunostained with streptavidin-AlexaFluor and analyzed by a BD FACSCalibur flow cytometer. (<b>C</b>) CACO-2/TC7 cells, either unstimulated or stimulated with biotinylated p31–43 (50 µg/ml), p10-mer (50 µg/ml) + biotinylated p31–43 (50 µg/ml), were immunostained with streptavidin-AlexaFluor. The images were acquired using an Olympus U RFL fluorescence microscope.</p

Inhibitory effect of gliadin peptide 10-mer on IRAK1 phosphorylation and NF-kB activation.

<p>CACO-2/TC7 cells, either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml), were analyzed by Western blot for IRAK1 phosphorylation and NF-kB activation. (<b>A</b>) Phosphorylated levels of IRAK1 (p-IRAK1) were analyzed in whole cell extracts by Western blot with anti-phospho-IRAK1 antibodies; for control, the blotted membranes were stripped and reprobed with anti-IRAK-1 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>B</b>) NF-kB activation was analyzed in whole cell extracts by Western blot with anti-phospho-NF-kB p65 Ser antibodies; for control, the blotted membranes were stripped and reprobed with anti-NF-kB p65 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software and peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments.</p

Inhibitory effect of gliadin peptide 10-mer on cytokine production.

<p>CACO-2/TC7 cells either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml) were analyzed for cytokine production. Cell supernatants were analyzed for pro-inflammatory cytokines IL-6 (<b>A</b>) and IL-8 (<b>B</b>) release by an ELISA kit. Statistical analysis: *P<0.01 <i>versus</i> control; §P<0.05 <i>versus</i> PT-Gly. Results are expressed as mean ± SD; n = 3.</p

Inhibitory effect of gliadin peptide 10-mer on ERK and p38 MAPK phosphorylation.

<p>CACO-2/TC7 cells either unstimulated or stimulated with PT-Gly (1mg/ml), p10-mer (50 µg/ml), p10-mer (50 µg/ml) + PT-Gly (1mg/ml), were analyzed by Western blot for ERK and p38 phosphorylation. Phosphorylated levels of ERK were analyzed in whole cell extracts by Western blot with anti-phospho-ERK1/2 antibodies; for control, the blotted membranes were stripped and reprobed with anti-ERK1/2 antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. (<b>B</b>) Phosphorylated levels of p38 MAPK were analyzed in whole cell extracts by Western blot with anti-phospho-p38 MAPK antibodies; for control, the blotted membranes were stripped and reprobed with anti-p38 MAPK antibodies. Bound antibodies were visualized with HRP-conjugated IgG and immunoreactivity was assessed by ECL. Densitometric analysis was performed using ImageJ version 1.46 software and peaks were reproduced by reading the Western Blot bands. One example representative of 3 experiments.</p