function 7 block  (MathWorks Inc)

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: 17β-E2 stimulation of human SKBR3 breast cancer cells promotes morphological alterations and enhanced FN matrix assembly. SKBR3 cells were allowed to adhere to glass coverslips in FN-reduced serum and then left unstimulated or treated with 17β-E2 (10 nm) for 2 h in the absence (A–J) or presence (K–U) of human FN (hFN) (2 μg/ml) and then fixed. Changes in cellular morphology were visualized by differential interference contrast (DIC) microscopy. Integrin α5β1 was detected using the integrin α5β1 inhibitory mAB, SAM-1 and Alexa 488-coupled antimouse IgG. Surface-associated hFN and emergent fibrils were visualized with hFN-specific rabbit polyclonal antibodies and Alexa 594-coupled antirabbit IgG. Colocalization of hFN and integrin α5β1 and an overlay of hFN and DIC are shown. The white arrow illuminates confluence of hFN and integrin α5β1 in samples receiving no exogenous FN. Bar, 10 μm.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Microscopy

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: Concentration of FN-occupied integrin α5β1 at matrix adhesions in response to estrogen stimulation. SKBR3 cells were allowed to adhere to glass coverslips in FN-reduced serum, and then left unstimulated (A, C, E, and G) or treated with 17β-E2 (10 nm) (B, D, F, and H) for 2 h in the presence of hFN (2 μg/ml) and then fixed. Changes in cellular morphology were visualized by differential interference contrast (DIC) microscopy. Conformer-specific integrin α5β1 mAB (SNAKA51) was employed to identify FN-occupied integrin α5β1 and was visualized using Alexa 488-coupled antimouse IgG. FN was detected using hFN-specific rabbit antibodies and Alexa 594-coupled antirabbit IgG. Colocalization of hFN and FN-occupied integrin α5β1 and an overlay of hFN and DIC are shown. Bar, 10 μm.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Concentration Assay, Microscopy

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: Integrin α5β1 is a necessary signaling intermediary for GPR30-dependent EGFR transactivation. ErbB1 tyrosyl phosphorylation was measured in MDA-MB-231 (GPR30) cells that were left untreated (UN) or pretreated with: (A) function blocking integrin-specific mABs for α5β1 (mAB P1D6; 10 μg/ml) or α2β1 (mAB AK-7; 20 μg/ml) before stimulation or (B) soluble RGD or RGE peptides (1 mm) and then stimulated with E2 or EGF. Tyrosyl phosphorylation of immunoprecipitated erbB1 was determined by immunoblotting with phosphotyrosine-specific mAB, 4G10. Reprobing with sheep EGFR antibodies assessed ErbB1 recovery.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Blocking Assay, Immunoprecipitation, Western Blot

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: Restoration of integrin β1 expression in integrin β1-null mouse epithelial cells is necessary for GPR30-induced erbB1 tyrosyl phosphorylation and FN fibril formation. Mouse GE11 (integrin β1 knockout) epithelial cells or GE11 cells stably transfected with integrin β1 subunit protein (GE11β1) were assessed for their capacity to promote estrogen-mediated (A) erbB1 tyrosyl phosphorylation or (B) FN matrix assembly. Cells were made quiescent and then left untreated (Un) or stimulated with 17β-E2 (1 nm), ICI 182,780 (1 μm), or EGF (10 ng/ml). FN matrix assembly (18 h) and EGFR transactivation (15 min) were measured as described in Fig. 3​3.. C, GE11β1 cells were stably transfected with HA-GPR30Δ154 Hygro (B, D, F, and H) or vector, pcDNA3.1(+) Hygro (A, C, E, and G) and then treated with 17α-E2, 17β-E2, ICI 182,780, or ATII for 18 h in the presence of hFN (2 μg/ml). Fibril formation was assessed as in Fig. 2​2.. Insets depict expanded views of fibrillogenesis. Bar, 10 μm.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Expressing, Knock-Out, Stable Transfection, Transfection, Plasmid Preparation

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: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

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: Engagement of integrin α5β1 is required for GPR30-mediated, Shc-integrin α5β1 protein complexes. SKBR3 cells transfected with mutant GST-317Y/F-Shc, GST-WT-Shc, or GPR30Δ154 received no peptide, RGD peptide, or control RGE peptide and then were left untreated or stimulated with 17β-E2 or EGF for 15 min and lysed in Triton X-100. Shc-integrin α5β1 complexes were determined as described above. The relative amount of integrin α5β1-associated recombinant Shc protein was computed from densitometric tracings from two experiments and normalized relative to the amount of total integrin α5β1.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Transfection, Mutagenesis, Recombinant

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: Model depicting the role of integrin α5β1 and Shc in FN matrix assembly and EGFR transactivation. Estrogen action via the 7TMR GPR30 results in Gβγ-subunit protein-dependent activation of integrin α5β1 as measured by Shc protein coupling and FN matrix assembly. Activated integrin α5β1, in turn, promotes erbB1 transactivation by release of membrane-tethered HB-EGF, resulting in EGFR tyrosyl phosphorylation (PY). Data provided here indicate that activation of integrin α5β1 is a prerequisite event for EGFR transactivation by GPR30 and that sequestration of pro-HB-EGF or inhibition of the tyrosine kinase activity of erbB1 does not interfere with GPR30-mediated FN matrix assembly.

Article Snippet: The following antibodies were purchased as indicated: sheep polyclonal EGFR antibody from Millipore Corp. (Milford, MA); erbB1-specific mAB, Ab-1, from EMD Biosciences (San Diego, CA); phosphotyrosine-specific mAB, 4G10, from Upstate Biotechnology, Inc.; inhibitory integrin α5β1 mAB SAM-1 from Chemicon; rabbit polyclonal antibodies (AB1949) specific for the cytoplasmic tail of integrin α5 subunit protein from Chemicon; mAB clone 2, against the N terminus of integrin α2 (611017) and mAB clone 1, against the C terminus of integrin α5 (610633) from BD Transduction Laboratories (Lexington, KY); function-blocking integrin mABs specific for integrins α2β1 (AK-7) and integrin α5β1 (P1D6) from EMD Biosciences.

Techniques: Activation Assay, Inhibition, Activity Assay

Journal:

Article Title: STAT3 SIGNALING IS INDUCED BY INTERCELLULAR ADHESION IN SQUAMOUS CELL CARCINOMA CELLS

doi: 10.1016/j.yexcr.2007.09.018

Figure Lengend Snippet: Localization of phosphorylated STAT3-Y705 during Ca2+-induced cell-cell contact. Immunofluorescence analysis of HSC-3 cells cultured to 100% confluence and subjected to Ca+2 switch assay. Cells were either left untreated or treated with EGTA for 45 min and then exposed to Ca+2-containing media for 60 min. Cells were then fixed, permeabilized and processed for immunostaining with antibodies to E-cadherin (red) and phosphorylated STAT3-Y705 (green); DAPI was used to stain nuclei (blue). Magnified images of the cell are indicated by the dashed boxes. Arrow indicates junctional localization of E-cadherin and phospho-STAT3-Y705. Bars, 20 μm; 5μm

Article Snippet: Function blocking anti-E-cadherin antibody, clone She-78-7 was from Zymed Laboratories (San Francisco, CA).

Techniques: Immunofluorescence, Cell Culture, Immunostaining, Staining

Journal:

Article Title: STAT3 SIGNALING IS INDUCED BY INTERCELLULAR ADHESION IN SQUAMOUS CELL CARCINOMA CELLS

doi: 10.1016/j.yexcr.2007.09.018

Figure Lengend Snippet: Inhibition of cell-cell contact by function-blocking E-cadherin antibody inhibits STAT3 phosphorylation. (A) Serum-starved confluent HSC-3 cells were processed for Ca2+-switch assay in presence of control IgG or function blocking E-cadherin antibody (She78-7) for 30 or 60 min. Cell lysates were prepared and analyzed for phosphorylated STAT3-Y705 and total STAT3. (B) HSC-3 cells were detached non-enzymatically, permitted to form MCAs in the presence of 5 μg/ml E-cadherin functional blocking antibody or control isotype IgG. Phase-contrast microphotographs of cells after 6 h of MCA culture. Bar, 20 μm. (C) Corresponding STAT3 phosphorylation status is shown as assessed by immunoblotting with anti-pSTAT antibody of the cell extracts. Results are representative of three independent experiments.

Article Snippet: Function blocking anti-E-cadherin antibody, clone She-78-7 was from Zymed Laboratories (San Francisco, CA).

Techniques: Inhibition, Blocking Assay, Functional Assay, Western Blot