Nodal expression and effects of Nodal blocking antibody on human breast cancer cells. In (A) immunofluorescence staining confirms Nodal expression in a subpopulation of MDA-MB-231 and MDA-MB-468 human breast cancer cells (bar = 20 μm). Western blotting of conditioned media from MDA-MB-231 and MDA-MB-468 reveals a band in each cell line (red box) corresponding to the molecular weight of pro-Nodal (approximately 36 KD) (B). Treatment of human breast cancer cell lines MDA-MB-231 and MDA-MB-468 with Nodal blocking antibody causes a dose dependent reduction in Nodal and P-Smad2 expression, as determined by Western blot analysis, with the most significant reduction observed using 4 μg/ml of Nodal blocking antibody (C). (*P < 0.05) (OD unit +/- SD = densitometric units +/- standard deviation).