Figure 3
β3 Integrin overexpression in NMuMG cells. NMuMG cells were engineered by bi-cistronic retroviral transduction to stably express GFP alone (control), WT β3 integrin (WT) and GFP, or its inactive mutant D119A and GFP. (a) Detergent-solubilized whole cell extracts (50 μg/lane) were prepared to analyze expression of the αv and β3 integrin subunits, and of TβR-II. Differences in protein loading were monitored by immunoblotting ERK1/2, whose expression in NMuMG cells was not altered by TGF-β treatment. (b) The percentage of NMuMG cells expressing human recombinant β3 integrin or endogenous αv integrin was determined by FACS analysis of cells incubated with either anti-human β3 integrin or anti-mouse αv integrin antibodies. Histograms depict β3 and αv expression only in GFP-positive NMuMG cells and are from a representative experiment that was repeated twice with similar results. (c) Iodinated TGF-β1 was bound and chemically cross-linked to control (GFP), WT, and D119A β3 integrin expressing cells. Cytokine:receptor complexes were isolated by immunoprecipitation with anti-TβR-II antibodies, fractionated through 7.5% SDS-PAGE gels, and immobilized electrophoretically to nitrocellulose membranes. The upper panel shows a representative phosphor image of iodinated TGF-β1 bound to TβR-I and TβR-II. The middle panel shows that β3 integrin was present in isolated iodinated TGF-β1:receptor complexes as detected by anti-β3 integrin Western blot analysis of anti-TβR-II immunocomplexes. Differences in protein loading were monitored by probing detergent-solubilized whole cell extracts (50 μg/lane) with anti-ERK1/2 antibodies (bottom panel). BXL, binding and cross-linking; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; WT, wild type; TβR, TGF receptor; TGF, transforming growth factor; WCE, whole cell extracts.