Fig. 2. Global SMAD1/5 phosphorylation is increased under differentiation conditions and is evident in mitotic cells in the undifferentiated state. (A) Immunofluorescence microscopy of BGN2 hESCs in the undifferentiated and differentiated state. BGN2 cells were cultured for 5 days in CM, in nCM, in CM supplemented with 25 ng/ml BMP4 (as a control for SMAD1/5 activation) and in nCM supplemented with 2 µM BIO. Cells were decorated with an antibody specific for phosphorylated SMAD1/5, as well as SytoxGreen nuclear counterstain. Arrows indicate mitotic cells. Scale bars: 50 µm. (B) Western blot analysis of colcemide synchronized BGN1 hESCs. Cells were blocked at metaphase by incubation with 100 ng/ml demecolcine solution and harvested at 15 minutes and 4 hours post-release. Cells grown in CM alone and CM supplemented with 25 ng/ml BMP4 were used as controls for asynchronous and SMAD1/5-activated cells, respectively. Membranes were probed with antibodies specific for phosphorylated (P) SMAD1/5, SMAD1/5, phosphorylated Ser CDKs substrate and -tubulin (as a control for protein loading). (C) Immunofluorescence microscopy of blastocyst stage embryo containing mitotic cells. Mouse blastocyst embryos were fixed and labeled with anti-phospho SMAD1/5 antibody and SytoxGreen nuclear counterstain, and then imaged by confocal microscopy. Scale bars: 20 µm.