Skip to main content
Figure 7 | Breast Cancer Research

Figure 7

From: β3Integrin and Src facilitate transforming growth factor-β mediated induction of epithelial-mesenchymal transition in mammary epithelial cells

Figure 7

Src inhibition blocks TβR-II tyrosine phosphorylation in NMuMG cells. (a) NMuMG cells were pretreated for 60 min with either PP2 (10 μmol/l) or SU6656 (10 μmol/l). Afterward, the cells were stimulated with TGF-β1 (5 ng/ml) for 20 min, and subsequently were lysed and immunoprecipitated (1 mg/tube) with anti-TβR-II antibodies. The resulting immunocomplexes were used to phosphorylate recombinant GST-Smad3, which was visualized by immunoblotting with anti-phospho-Smad2 antibodies. Data are from a representative experiment that was repeated three times with similar results. (b) Control or β3 integrin expressing NMuMG cells were stimulated with TGF-β1 (5 ng/ml) in the absence or presence the Src inhibitor PP2 (10 μmol/l), as indicated. Afterward, detergent-solubilized whole cell extracts were prepared, immunoprecipitated, and subjected to Western blot analysis either with anti-phosphotyrosine (2 mg WCE/tube) or anti-TβR-II (1 mg WCE/tube) antibodies as indicated. Differences in protein loading were monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-ERK1/2 antibodies (bottom panel). (c) WT β3 integrin expressing NMuMG cells were transiently transfected with cDNAs encoding either constitutively active (CA) or dominant negative (DN) Src. Afterward, detergent solubilized whole cell extracts were prepared, immunoprecipitated with anti-phosphotyrosine (1 mg WCE/tube), and subjected to WB analysis with anti-TβR-II antibodies. Total Src expression was monitored by probing detergent solubilized whole cell extracts (50 μg/lane) with anti-Src antibodies, whereas exogenous Src expression was confirmed by reprobing stripped membranes with anti-Myc antibodies. Differences in protein loading were monitored by probing stripped membranes with anti-TβR-II antibodies. ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; IP, immunoprecipitated; PY, phosphotyrosine; TβR, TGF-β receptor; TGF, transforming growth factor; WB, Western blot; WCE, whole cell extracts; WT, wild type.

Back to article page