Journal:

Article Title: Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30

doi: 10.1210/me.2008-0262

Figure Lengend Snippet: GPR30-mediated FN matrix assembly occurs independently of EGFR transactivation in SKBR3 breast cancer cells. A, SKBR3 cells were fed rat FN (rFN) (25 μg/ml) in the presence of either 17β-E2 (1 nm) or ICI 182,780 (1 μm) or no agonist (untreated, UN) for 18 h. Alternatively, cells were pretreated with the pan MMP-inhibitor, GM6001 (10 μm for 15 min), Src-like kinase inhibitor, PP1 (50 nm for 15 min), or CRM-197 (200 ng/ml for 1 h) before receiving rFN in the presence of 17β-E2. Equivalent amounts of DOC-soluble or -insoluble protein were blotted with rFN-specific mAB IC3. The immunoblot shown is a representative of four independent experiments. Band intensities from individual experiments were measured using Scion Image software. Mean integrated optical densities (±sem) were measured for unstimulated (28.6 ± 11.5) and 17β-E2-stimulated (148.1 ± 15.7) cells. B, SKBR3 cells were pretreated with either the erbB1-specific inhibitor, tyrphostin AG1478 (1 nm) or vehicle (DMSO) for 15 min before receiving rFN in the presence of 17β-E2 (1 nm), ICI 182, 780 (1 μm), EGF (10 ng/ml), HB-EGF (10 ng/ml), or no agonist. FN matrix assembly was measured after 18 h of treatment as described above. C, SKBR3 cells treated with AG1478 or vehicle as described in panel B in the absence of exogenous rFN. Cells were then lysed and erbB1 tyrosyl phosphorylation was assessed in erbB1 immunoprecipitates that were immunoblotted with phosphotyrosine-specific 4G10 mAB. ErbB1 recovery was determined by reprobing the same filter with sheep EGFR antibodies. DMSO, Dimethylsulfoxide.

Article Snippet: Src-family tyrosine kinase inhibitor, PP1, was purchased from AG Scientific Inc. (San Diego, CA).

Techniques: Western Blot, Software, Phospho-proteomics

Journal:

Article Title: Coordinate Regulation of Estrogen-Mediated Fibronectin Matrix Assembly and Epidermal Growth Factor Receptor Transactivation by the G Protein-Coupled Receptor, GPR30

doi: 10.1210/me.2008-0262

Figure Lengend Snippet: GPR30 signaling promotes the formation of Src-dependent, Shc-integrin α5β1 protein complexes. Triton X-100 extracts prepared from MDA-MB-231 cells stably expressing vector or GPR30 were left untreated (UN) or stimulated with 17β-E2, EGF, or pretreated with the Src family kinase inhibitor, PP1 (50 nm; 15 min) before stimulation with 17β-E2. Integrin α5β1 or α2β1 was immunoprecipitated with rabbit antibodies specific for the integrin α5 or α2 subunit cytoplasmic tails, respectively. Associated Shc proteins were detected with Shc mAB, PG797. Recovery of integrin α5β1 or α2β1 was determined by blotting with integrin α5 or α2 subunit protein antibodies. Relative positions of endogenous p66, p52, and p46 Shc are designated.

Article Snippet: Src-family tyrosine kinase inhibitor, PP1, was purchased from AG Scientific Inc. (San Diego, CA).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation