A sensitive detection of phospho-Smad1/5/8 and Smad2 in Western blot analyses

The transforming growth factor-β (TGF-β) family is involved in a variety of physiological processes, and transmits signals through phosphorylation of Smad by the receptor complexes. In the present study, effects of blocking solution in Western blot analyses on detection of phosphorylated Smad1/5/8 and Smad2 were examined. When EzBlock was used as a blocking reagent, phosphorylated Smad1/8 and Smad2 were most efficiently detected. The anti-phospho-Smad2 antibody specifically recognized the phosphorylated form of Smad2, whereas the anti-phospho-Smad1/5/8 antibody also reacted to the unphosphorylated form. These antibodies did not react with the other Smads.