Detection of Smad4 in breast cancer cell lines, tumor tissues and surrounding normal breast epithelia using the monoclonal antibody clone B-8. (a) Detection of Smad4 in dilution series of total protein lysates of breast cancer tissue and MCF7 cells by dot blot. (b) Detection of Smad4 expression in Smad4 negative MDA468 cells, non malignant, immortal MCF10A cells, and Ras-transformed, increasingly malignant MCF10A derived cell lines by Western blot. Immunohistochemical detection of Smad4 in breast cancer cell lines (c: MDA 468; d: MCF10CA1a), breast carcinoma (e-h, j,k), and surrounding normal breast epithelia (i). Staining intensity varied between specimens: none (c,e), weak (f), moderate (d,g), and strong (h). Cytoplasm as well as nuclei stained positive (d,f-h). Within the same specimen tumor cells stained less than surrounding normal epithelia. (i) Lobular (closed arrowhead) and ductal (open arrowhead) breast epithelium, (j) lobular breast epithelium (closed arrowhead) and lobular breast carcinoma (open arrowhead), (k) ductal breast epithelium (closed arrowhead), ductal breast carcinoma (open arrowhead) and ductal breast carcinoma in situ (arrow).