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Figure 2


Fig. 2. Identification of the endofin interaction domain in Smad1 and PP1c. (A) Schematic representation of wild-type and mutant endofin. Three mutants were generated to disrupt these conserved domains: FYVE domain (C753S), Smad-binding domain (deletion of amino acids 814-860) and PP1c-binding domain (F872A). (B) Deletion of Smad-binding domain in endofin diminishes its interaction with Smad1. COS1 cells were transfected with Flag-Smad1, HA-tagged endofin and its mutants. Immunoblots in the two lower panels show the protein expression levels of the COS1 cell lysates. The upper panel shows anti-HA immunoprecipitates probed with anti-Flag antibody. (C) Co-IP of PP1c with endofin and the PBD(F872A) mutant. HA-tagged endofin and its mutant with disrupted PP1c-binding domain were co-transfected with PP1c into COS1 cells. The lysate was subject to immunoprecipitation with anti-HA. Immunoblots indicating the protein expression levels of the lysates of COS1 cells are shown in the two lower panels. The upper panel shows anti-HA immunoprecipitates probed with anti-Flag antibody.





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