The mode of action underlying halofuginones effect on Smad3 phosphorylation isn’t clear. In this research, we show for the very first time that halofuginone causes the phosphorylation of MAPK/ERK and Akt and encourages their association with Smad3 in cultured myoblasts and myotubes. The kinetics of this organization coincided with the reduction in Smad3 phosphorylation, and the addition of inhibitors which block either Akt or MAPK/ERK phosphorylation avoided the reduction in phosphorylation, indicating the particular role of those pathways in mediating Ibrutinib Src inhibitor halofuginones inhibitory effect on Smad3 signaling. While our studies in myoblasts and myotubes agree with studies demonstrating an role for phosphorylated Akt on Smad3 signaling in other cells, the role of MAPK/ERK in mediating the TGFB signaling pathway is less obvious. Some studies show that TGFB triggers MAPK/ERK phosphorylation, which promotes TGFB responses, while others report that MAPK/ERK pathway activation by ligands other than TGFB, o-r by overexpression of activated molecules upstream of ERK, disrupts Smad3 activation. Our results suggest that in muscle, MAPK/ERK is activated by halofuginone alone of TGFB, and may therefore play a part as a regulator of Endosymbiotic theory Smad3 phosphorylation. This is supported by: halofuginonedependent induced of MAPK/ERK phosphorylation in muscle cells and obstruction of this phosphorylation by a inhibitor, and the inhibitory influence of halofuginone on Smad3 phosphorylation on elements Ser423/425, identified by the antibody to phospho Smad3 used in this study. Since this receptor is not affected by halofuginone, this inhibitory influence was probably not mediated by the downregulation of TGFBRI, proven to phosphorylate these amino acids. Taken together, we suggest that part of the process through which halofuginone inhibits Smad3 signaling in muscle is via its association with MAPK/ERK and Akt. This process may not be exclusive to muscle cells since similar results were observed in an cell line and major cultures of muscle derived fibroblasts. It will be noted that other things, including the contribution of Smad7?which is upregulated by map kinase inhibitor halofuginone in epithelial cells?cannot be eliminated. Other signaling pathways, including the amino acid starvation result, have been recently proved to be triggered by halofuginone in order to prevent inflammatory T cell differentiation. Apparently, whereas the MEK inhibitor UO126 had no influence on Akt phosphorylation, the PI3K inhibitor Wortmannin did restrict halofuginone induced MAPK/ERK phosphorylation. Earlier studies demonstrate that PI3K inhibitors block activation of-the Raf/MEK/ERK process and that PI3K mediated PDK1 phosphorylates Ser222 and Ser226 on MEK1/2, respectively.