Western blot analysis revealed that Smad3 and Krüppel-like factor 6 (KLF6) are co-expressed in C2C12 myoblasts (a). Myogenin was used as a protein marker for differentiation and actin was used as a loading control. Pharmacological manipulation of the Transforming growth factor β (TGFβ) signaling pathway reveals that TGFβ regulates KLF6 protein expression through Smad3 but not MEK/ERK MAPK. (b) Western blot analysis indicates that 2 ng/ml TGFβ treatment elevates KLF6 protein expression and that this effect is abrogated in the presence of 5 μM of specific inhibitor of Smad3, Sis3. TGFβ treatment also inhibited the myogenic differentiation marker, myogenin protein expression level, and this effect was not abrogated by Sis3. (c) Western blot analysis revealed that TGFβ treatment enhances KLF6 expression through Smad3 but not ERK1/2 MAPK and that TGFβ treatment repressed myogenic differentiation through ERK1/2 MAPK but not Smad3; 10 μM U0126 was used as an inhibitor of the MEK/ERK MAPK pathway, 5 μM Sis3 was used for Smad3 inhibition and 2 ng/ml TGFβ were all used as indicated. Actin was used as a loading control.