TI - MAB21L2 interacts with SMAD1 in vitro and in vivo . AB - The antagonistic interaction between Mab21l2 and BMP4 may be either interpreted as evidence of a molecular interaction between MAB21L2 and some BMP signaling transducer , or alternatively suggest that MAB21L2 and some component of the BMP signaling pathway compete for a common cofactor . It is well established that the DNA -binding protein SMAD1 , once PHOSphorylated at the plasma membrane by the BMP receptor heterotetramer , assembles with the receptor -independent protein SMAD4 , enters the nucleus , establishes interactions with various cofactors , and acts by regulating the transcription of a host of target genes [reviewed in [22] ] . Because vertebrate MAB-21 proteins are mainly or exclusively distributed in the nucleus [6] , we investigated the possibility of a molecular interaction between MAB21L2 and SMAD proteins . In order to obtain in vitro evidence of this interaction , P19 cells were either treated with BMP4 or left untreated , and subsequently lysed . P19 cell extracts were then incubated with a resin-bound His-ZZ-tagged form of MAB21L2 , or with a control His-ZZ-coupled resin . Eluates were analyzed by immunoblotting with a polyclonal anti-SMAD1 antibody . The experiment revealed a clear interaction between synthetic His-ZZ-MAB21L2 and endogenous SMAD1 that was absent when P19 lysates were incubated with the control resin (Figure 5A) . Although BMP4 treatment seemed to enhance it considerably , the interaction was not entirely BMP4 -dependent , suggesting that conformational changes secondary to receptor -mediated PHOSphorylation may not be strictly required for SMAD1 to interact with MAB21L2 . These results prompted more questions , such as whether the interaction between MAB21L2 and SMAD1 is direct and whether it is dependent on prior formation of a SMAD1-SMAD4 complex . Once again , we resorted to affinity chromatography , incubating in vitro-translated , 35S-Met-labeled SMAD1 and SMAD4 with a resin containing His-ZZ-MAB21L2 . This experiment suggested that the interaction with SMAD1 is direct , and that MAB21L2 does not assemble with SMAD4 directly but only through SMAD1 , indicating that the formation of a SMAD4-MAB21L2 complex is mediated by SMAD1 (Figure 5B) . Indirectly , this experiment also showed that MAB21L2 does not obviously compete with SMAD4 to assemble with SMAD1 . Finally , we investigated whether the in vitro interaction observed between MAB21L2 and SMAD1 could be replicated in vivo . P19 cells co-transfected with myc-MAB21L2 and flag-SMAD1 were either treated with BMP4 or left untreated . In parallel , Xenopus laevis embryos were injected at the four cell stage into the animal pole of each blastomere with myc-Mab21l2 and flag-Smad1 mRNAs , and coinjected with either BMP4 mRNA or H2O . The embryos were grown until mid-gastrula stage ( st 105/11 ) . Both cell - and embryo lysates were subjected to immunoprecipitation , using an anti flag monoclonal antibody , whereas an anti-myc antibody was utilized for immunodetection in Western blotting . Experiments conducted in transfected P19 cells showed that SMAD1 coprecipitates with MAB21L2 , and that the interaction is enhanced by activation of BMP signaling , which is required for nuclear localization of receptor -dependent SMADs (Figure 5C) . Likewise , MAB21L2-SMAD1 co-precipitation took place especially , albeit not exclusively , in BMP4-coinjected embryos ( Figure 5D ) . Interaction of the two proteins in the absence of exogenous BMP4 can likely be explained by the abundance of endogenous BMP2/4 ligands in stage 11 gastrulae .