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Figure 3 | Breast Cancer Research

Figure 3

From: Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-β and Neu pathway signaling studies: identification of a novel model for TGF-β-induced epithelial-to-mesenchymal transition

Figure 3

Growth response of the three cell lines to recombinant human epidermal growth factor (hEGF), heregulin (HRG)-β1, and transforming growth factor (TGF)-β1. [3H]thymidine incorporation measured in BRI-JM01, BRI-JM04, and BRI-JM05 cell lines after 24 hours of exposure to serial dilutions of (a) hEGF, (b) HRG-β1, or (d) TGF-β1. Experiments were conducted in quadruplicate and repeated three times with similar results (for details see text). (c) Western blot analysis of whole cell lysates (20 μg total protein/lane) evaluating ErbB-1, ErbB-2, ErbB-3, and ErbB-4 expression. Additional ErbB-2/Neu immunoprecipitations showed this receptor to be highly phosphorylated in the BRI-JM04 and BRI-JM05 cells. (e) Affinity cross-link labeling of the TGF-β type I, II, III receptors expressed on the BRI-JM01, BRI-JM04 and BRI-JM05 cells using [125I]TGF-β1 in the absence (-) or presence (+) of 100× excess of unlabeled TGF-β1. Enzymatic deglycosylation using both heparinitase I/II/III and chondroitinase ABC collapses the diffusely migrating TGF-β type III receptor band to a core protein (approximately 115 kDa, indicated as TβRIII**) labeled with either [125I]TGF-β1 or [125I]TGF-β2. TGF-β responsive Mv1Lu cells served as a positive control in the TGF-β growth assay and the cell surface cross-link affinity labeling studies. (f) RT-PCR analysis of BRI-JM01 mRNA using two different primer sets (set 1, set 2) confirmed the presence of TGF-β type III receptor transcripts in the BRI-JM01 cells. TGF-β type III receptor-expressing BRI-JM05 cells (part e) were used as a positive control for TGF-β type III receptor expression, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Experiments were repeated twice with similar results.

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