TI - PHOSphorylated Smad2 co-localises with MEF2A in the nucleus of C2C12 mt . AB - It is likely that , as in other cellular systems , TGF-beta receptors are activated by TGF-beta and are able to PHOSphorylate both Smad2 and 3 , allowing them to bind to Smad4 and migrate to the nucleus . In contrast , Smad1 is not TGF-beta sensitive . To determine the cellular localisation of Smad proteins at different stages of muscle development , cells were immunostained with isoform-specific antibodies . Endogenous MEF2A , Smad1 , PHOSphorylated Smad2 and 3 were detected in mt in the presence of exogenous TGF-beta ( Fig 5 ) . MEF2A is strongly expressed in the nuclei of mt , and the addition of TGF-beta does not alter either localisation or expression levels of MEF2A . In contrast , Smad1 remained cytoplasmic in both the absence and presence of TGF-beta . Interestingly , however , we observed differential regulation of Smad2 and 3 in differentiating mt . Without the addition of exogenous TGF-beta , PHOSphorylated Smad2 translocated to the nucleus in differentiating mt . Conversely , Smad3 remained cytoplasmic in differentiating mt , but did localise to the nucleus when cells were treated with TGF-beta . These data suggest that Smad2-MEF2A cooperativity may exist in later stages of muscle development . These data also suggest that Smad2 and 3 are differentially regulated during muscle cell differentiation .