Expression levels of TGF-β1 were determined by ELISA (A). Kidney tissue lysates collected after 3 weeks of daily feeding of the mixture of adenine and potassium oxonate with or without 3-MA administration were prepared and subjected to immunoblot analysis with antibodies to TGF-βRІ, p-Smad3, Smad3, and GAPDH (B). Expression levels of TGF-βRІ were quantitated by densitometry and normalized with GAPDH (C). Expression levels of p-Smad3 and Smad3 were calculated by densitometry and the ratio between p-Smad3 and Smad3 was determined (D). Kidney tissue lysates were prepared and subjected to immunoblot analysis with antibodies to p-TAK1, TAK1, and GAPDH (E). Expression levels of p-TAK1 (F) and TAK1 (G) were quantitated by densitometry and normalized with GAPDH. Data are represented as the mean ± S.E.M. Means with different superscript letters are significantly different from one another (P<0.05).