TI - Figures and Tables . AB - Figure 1 TGF-beta responsive co-modulators , Smad2 and 4 , increase the activities of both GAL4-MEF2A and GAL4-MEF2C. ( A and B ) C2C12 cells were co-transfected with pCMV5B-TbetaRI (T204D) , pCMV5B-Smad2 , pCMV5B-Smad4 , 5xGAL4-luc , GAL4 (DBD) , GAL4-MEF2A , GAL4-MEF2C and pSV-betagal as indicated and treated with ( + ) or without ( - ) 2 ng/ml of TGF-beta in 1% FBS . The transcriptional activation domains of both MEF2 isoforms are fused to the DNA binding domain ( DBD ) of GAL4 to form GAL4-MEF2A ( 91-507 amino acids ) and GAL4-MEF2C ( 87-442 amino acids ) . GAL4 (DBD) contains the GAL4 DBD only . The reporter plasmid , 5xGAL4-luc , contains five copies of the GAL4 binding site linked to the adenovirus EIB promoter and the firefly luciferase gene . After 72 h , beta-galactosidase activities were measured and used to normalise luciferase activity values . Two or more sets of assays were performed in both COS and C2C12 cells with comparable results . Each data point is a mean of triplicate samples from single experiments and the error bars represent the standard error of the mean ( SEM ) . Figure 2 In C2C12 cells , the activities of both GAL4-MEF2A and GAL4-MEF2C are enhanced by the over-expression of Smad2 and 4 with wild-type TGF-beta receptors ( TbetaR-I and -II ) , the presence of a TGF-beta dominant negative receptor , TbetaRII (K277R) . ( A-D ) C2C12 cells were co-transfected with pCMV5B-TbetaRII (K277R) , pCMV5B-TbetaR-I , pCMV5B-TbetaR-II , pCMV5B-Smad2 , pCMV5B-Smad3 , pCMV5B-Smad4 , 5xGAL4-luc , GAL4 (DBD) , GAL4-MEF2A ( 91-507 ) , GAL4-MEF2C ( 87-442 ) and pSV-betagal as indicated . Twenty-four hours after transfection , growth medium was replaced with 1% FBS + - 2 ng/ml of TGF-beta . Cells were harvested 72 h after transfection for luciferase assays . beta-galactosidase activities were used to normalise for transfection efficiency . Two or more sets of experiments were performed with comparable results . Each data point is the mean of triplicate samples from single experiments and the error bars represent the SEM . Figure 3 A physical interaction between endogenous Smad2 and MEF2A in C2C12 mt was detected through co-immunoprecipitation. ( A and B ) COS cells were transiently transfected with pCMV5B-TbetaRI (T204D) , pCMV5B-Smad2 , pCMV5B-Smad4 and pMT2-MEF2A as indicated . Cell lysates were immunoprecipitated with antibodies raised against either MEF2A ( alpha-MEF2A ) or Smad2 (alpha-Smad2) . (A) An immunoblot probed by an alpha-Smad2 antibody contains : 15 ul of anti-MEF2A immunoprecipitate ( alpha-MEF2A ip ) in lanes 1 and 3 ; 15 ul of anti-Smad2 immunoprecipitate ( alpha-Smad2 ip ) in lanes 5 and 7 ; and 30 ug of C2C12 cell extract in lane 9 . The arrow indicates Smad2 protein . (B) An immunoblot probed by an alpha-MEF2A antibody contains : 15 ul of alpha-Smad2 ip in lane 1 ; 1 ul of alpha-Smad2 ip in lane 2 ; 20 ug of C2C12 cell extract in lane 4 . Lane 3 was empty ( C ) An immunoblot probed by an alpha-MEF2A antibody contains : a molecular weight marker in lane 1 ; 1 ul of alpha-MEF2A ip from myoblast lysate ( mb ) in lane 2 ; 1 ul of alpha-MEF2A ip from myotube lysate ( mt ) in lane 3 ; 20 ul of alpha-Smad2 ip from mb in lane 5 ; and 20 ul of alpha-Smad2 ip from mt in lane 6. ( D ) A negative control for the co-immunoprecipitation assays was performed . An immunoblot probed with an alpha-MEF2A antibody contains : 20 ul of alpha-mouse IgG ip from mt lysate in lane 1 ; 50 ug of mt cellular extract ( mt ) in lane 3 ; 1 ul of mt lysate in lane 5 ( used for alpha-Smad2 ip in lane 7 ) ; 20 ul of alpha-Smad2 ip from mt in lane 7 ; 15 ul of pre-cleared supernatant from alpha-Smad2 ip ( lane 7 ) in lane 9 ; and a molecular weight marker in lane 10 . Lanes 2 , 4 , 6 and 8 were empty. ( B-D ) The arrow indicates MEF2A protein. ( A-D ) The arrowheads indicate IgG recognised by the goat anti-rabbit secondary antibody . The bands below the IgG are non-specific. ( E ) C2C12 mb were plated at 25% confluence and 24 h later the medium was replaced with differentiation medium with ( + ) or without ( - ) 2 ng/ml TGF-beta . After 2 , 4 and 6 days (d) , protein expression was analysed by loading 20 ug of each extract on a SDS-polyacrylamide gel and western blotting using an anti-Smad2 antibody . Jurkats cell extract ( JC ) was used as a control . Figure 4 MEF2 proteins associate with GST-Smad2 in vitro. ( Top ) In a GST pull-down assay , in vitro translated [35S] methionine-labelled MEF2A ( 2 ul ) or MEF2C ( 25 ul ) was mixed with 5 ug of GST ( lanes 3 and 6 ) or GST-Smad2 ( lanes 4 and 7 ) immobilised on glutathione-agarose beads . After washing , 35S-labelled bound proteins were analysed by SDS-PAGE and autoradiography . Aliquots of 0.4 ul MEF2A ( lane 2 ) and 0.5 ul MEF2C ( lane 5 ) in vitro translation reactions were included as references . The arrows indicate the position of MEF2A or MEF2C. ( Bottom ) Coomassie blue staining of the gel shows that comparable amount of the GST or GST-Smad2 proteins were loaded . The open arrows ( lanes 3 , 4 , 6 and 7 ) indicate the position of the corresponding GST or GST-Smad2 fusion proteins . Figure 5 Localisation of MEF2A , Smad1 , Smad2 , PHOSphorylated-Smad2 and Smad3 in cultured muscle cells . C2C12 mb were grown in differentiation media in the absence of exogenous TGF-beta ( -TGF-beta ) until myotubes formed after 4 days ( left and middle panels ) . At this point , myotubes were either fixed for immunofluorescence or exposed to differentiation medium + 2 ng/ml TGF-beta ( +TGF-beta ) for 24 h ( right panels ) . Mt were fixed and immunostained using specific antibodies raised against MEF2A , Smad1 , Smad2 , PHOSphorylated Smad2 or Smad3 . Figure 6 MEF2A is not retained in the cytoplasm by TGF-beta signalling. ( A and B ) C2C12 mb were co-transfected with Flag-pCMVB-MADR2 (3SA) and cytomegalovirus-eGFP using lipofectamine . The expression of GFP was used to determine which cells had been transfected . Cells were fixed with methanol and immunostained using either an anti-Flag ( alphaFlag ) antibody or an anti-MEF2A antibody ( alphaMEF2A ) . Figure 7 The Smad2-MEF2 interaction is dependent on p38 MAPK PHOSphorylation sites in MEF2C and in mt , the endogenous MEF2 activity is increased by the addition of TGF-beta. ( A ) COS cells were co-transfected with pSV-betagal , pCMV5B-Smad2 , pCMV5B-Smad3 , pCMV5B-Smad4 , pCMV5B-TbetaRI (T204D) , GAL4 (DBD) , GAL4-MEF2C ( 87-442 ) , GAL4-MEF2C (S387A) , GAL4-MEF2C (T293,300A) and 5xGAL4-luc as indicated . The construct GAL4-MEF2C (S387A) contains a replacement of serine 387 for alanine and GAL4-MEF2C (T293,300A) contains the exchange of two threonines for alanines at positions 293 and 300 . Cells were harvested 72 h after transfection for luciferase assays. ( B and C ) C2C12 cells were co-transfected with pSV-betagal , pMT2 , pMT2-MEF2A , pMEF2-luc , GAL4-MEF2A , GAL4-MEF2C , GAL4 (DBD) and 5xGAL4-luc as indicated . Twenty-four hours later the medium was replaced with growth medium . One day later , growth medium was replaced with differentiation medium , which was replaced every 2 days . Mt formed after 4 days , at which point the medium was replaced with differentiation medium and either 2 ng/ml TGF-beta ( + ) or an equal volume of TGF-beta diluent ( - ) . Cells were harvested after 24 h for luciferase assays. (A-C) beta-galactosidase activities were used to normalise for transfection efficiency . Two or more sets of experiments were performed with comparable results . Each data point is a mean of triplicate samples from single experiments and the error bars represent the SEM .