Figure 6

Expression of TLR2 and TLR4 mRNA and protein in cultured BECs and effects of anti-TLR antibody on PAMPs-induced CDX2 and MUC2 expression. (a–f) BECs from PCK rats. (g) BECs from control rats. (a) TLR2 and TLR4 mRNA are expressed at the predicted size (245 and 205 bp, respectively) in cultured BECs under baseline conditions, as shown by RT-PCR. (b) Membranous and cytoplasmic expression of TLR2 and TLR4 is detected in cultured BECs (arrows). Immunocytochemistry for TLR2 and TLR4. (c and d) Preincubation with anti-TLR4 or TLR2 antibody (10 μg/ml) for 30 min reduced CDX2 and MUC2 mRNA expression induced by treatment with LPS (10 μg/ml) or synthetic lipopeptide Pam3 (10 μg/ml) for 6 h, respectively. Normal rabbit IgG (10 μg/ml) did not affect CDX2 or MUC2 expression induced by LPS or Pam3. RT-PCR for CDX2 and MUC2. (e and f) Preincubation with anti-TLR4 or TLR2 antibody (10 μg/ml) for 1 h reduced CDX2 and MUC2 protein expression induced by treatment with LPS (10 μg/ml) or Pam3 (10 μg/ml) for 72 h, respectively. Western blotting for CDX2 and MUC2. (g) Preincubation with anti-TLR4 antibody (10 μg/ml) for 30 min reduced CDX2 and MUC2 mRNA expression induced by treatment with LPS (10 μg/ml) in cultured BECs from normal rats as seen in PCK rats (Figure 6c). Normal rabbit IgG (10 μg/ml) did not affect CDX2 or MUC2 expression induced by LPS. RT-PCR for CDX2 and MUC2.