Effects of MTX and TGF-β on TGF-βR protein.

<p>Relative expression of TGF-βR protein in jejunum, as determined by Western blot analysis, of rats following MTX induced mucositis (A). Values are mean ± SEM. CONTR–control, MTX-methotrexate, TGF-β-transforming growth factor beta. *P<0.05 MTX-TGF-β and CONTR-TGF-β versus control rats, ^†P<0.05 MTX-TGF-β versus MTX rats. ERK was detected using anti-ERK antibody to verify equal protein loading. (<b>B</b>) Immunohistochemistry showing the expression of the type II TGF-β receptor along the crypt-villus axis in jejunum. Sections were incubated with an affinity purified rabbit anti-rat type II TGF-β receptor polyclonal antibody which is non-reactive with the type I receptor. Color was developed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045221#s2" target="_blank">Materials and methods</a>. There was no change in the expression of the receptor along the villus-crypt axis (arrows-expression in villus, arrowheads- expression in crypt) in control rats. Treatment with MTX resulted in a lower type II TGF-β2 receptor expression compared to control animals. Treatment of control and MTX animals with TGF-β2 resulted in a more significant receptor expression compared to control and MTX-non-treated animals, both in the crypt region and along the entire villus, especially at the basolateral side of enterocytes. The staining was more prominent in differentiated villus tip cells compared to crypt cells. The changes in distribution of Type II TGF-β2 receptor was correlated with the protein levels observed in the Western blot analysis.