Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Decreasing caveolin 1 expression attenuates 17β-estradiol-mediated activation of eNOS. PAEC were transfected with either a caveolin 1 siRNA or a scrambled siRNA (as a control). Immunoblot analysis demonstrates a significant decrease in caveolin 1 protein levels in caveolin 1 siRNA-transfected cells (A and B; *, P < 0.05 vs. scrambled siRNA). Plasma membrane fractions were isolated and subjected to immunoblotting with either an antibody against ERα or eNOS. Decreasing caveolin 1 expression results in a significant reduction in ERα localization to the plasma membrane (C and D; *, P < 0.05 vs. scrambled siRNA) although total ERα levels are unchanged (C). The siRNA-mediated knockdown of caveolin 1 does not alter eNOS localized to the plasma membrane (E and F), Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). The siRNA-mediated knockdown of caveolin 1 significantly inhibits 17β-estradiol-stimulated (100 nm, 30 min) eNOS phosphorylation at serine 1177 (G and H) and NO generation (I). The siRNA-mediated knockdown of caveolin 1 does not inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel J) or VEGF (100 ng/ml, 15min; panel K). Data are mean ± sem, n = 3–6. *, P < 0.05 vs. no 17β-estradiol; †, P < 0.05 vs. 17β-estradiol-treated scrambled siRNA.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Expressing, Activation Assay, Transfection, Western Blot, Isolation, Marker

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: 17β-Estradiol stimulates caveolin 1 phosphorylation through the activation of pp60Src. PAEC were exposed to pp60Src inhibitor, PP2 (10 μm, 30 min), before 17β-estradiol (100 nm, 30 min). 17β-Estradiol significantly increases phospho-Tyr14 caveolin 1, and PP2 inhibits this phosphorylation although total caveolin 1 levels are unchanged (A and B). 17β-Estradiol also produces significantly higher eNOS phosphorylation at serine 1177 compared with untreated cells, and pretreatment with the ER antagonist, ICI 182 780 (10 μm, 30 min) or PP2 significantly attenuates eNOS phosphorylation at serine 1177 (C and D). 17β-Estradiol treatment also induces a significant increase in NO generation, and pretreatment with ICI 182,780 or PP2 results in significant reduction in NO production (E). Data are mean ± sem n = 3–4. *, P < 0.05 vs. untreated; †, P < 0.05 vs. 17β-estradiol treated.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Activation Assay

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation is not required for the localization of ERα to the plasma membrane. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav) or a tyrosine 14-deficient mutant (AdY14FCav) indicates significant overexpression of AdWTCav and AdY14Cav (A and B). There is significantly higher ERα in the plasma membrane in both AdWTCav- and AdY14Cav-transduced cells (C and D). AdY14Cav overexpression did not alter eNOS localization to the plasma membrane (E and F). Immunoblotting with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C and E). Immunofluorescence analysis of cells transduced with AdGFP, AdWTCav-1, or AdY14FCav-1 were then transfected with an ERα-GFP construct. The plasma membrane of these cells was labeled with Alexa Fluor 594 WGA (red). The extent of membrane localization of ERα was then determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA and green fluorescence of ERα-GFP (G); the white bar in each individual panel represents 30 μm). The intensity of yellow fluorescence is significantly higher in cells transduced with either AdWTCav or AdY14Cav (H). Data are mean ± sem n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Western Blot, Transduction, Construct, Mutagenesis, Over Expression, Marker, Immunofluorescence, Transfection, Labeling, Fluorescence

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation enhances the interaction of ERα with PI3 kinase. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the interaction of ERα with caveolin 1 (A and C) or PI3 kinase (A and B) was determined. 17β-Estradiol exposure causes a dissociation of the ERα-caveolin 1 complex (A and C). This dissociation was enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1 (A and C). 17β-Estradiol also significantly increases the interaction of ERα with PI3 kinase (A and B). Transduction with AdWTCav potentiates the 17β-estradiol-mediated interaction (A and B) whereas AdY14FCav attenuates this effect (A and B). Data are mean ± sem; n = 3. *, P < 0.05 vs. untreated cells; †, P < 0.05 vs. AdGFP with 17β-estradiol. IB, Immunoblotting; IP, immunoprecipitation.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Transduction, Western Blot, Immunoprecipitation

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Caveolin 1 phosphorylation enhances Akt activation and NO signaling in response to 17β-estradiol. PAEC were transduced with AdGFP, AdWTCav-1, or AdY14FCav-1, exposed or not to 17β-estradiol, and the effect on Akt activity (A and B), eNOS phosphorylation at serine1177 (C and D), and NO generation (E) was determined. The 17β-estradiol-mediated increase in Akt activity (A and B), eNOS phosphorylation at serine 1177 (C and D), and NO generation (E) is enhanced in cells overexpressing wild-type caveolin 1 and attenuated in cells overexpressing Y14F caveolin 1. The overexpression of wild-type caveolin 1 (F and G) or AdY14F (H and I) did not significantly attenuate the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min) or VEGF (100 ng/ml, 15min). Data are mean ± sem; n =3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. AdGFP with 17β-estradiol.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Activation Assay, Transduction, Activity Assay, Over Expression

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: The scaffold domain of caveolin 1 is required for ERα trafficking to the plasma membrane but not for protein-protein interaction. Immunoblot analysis of PAEC transduced with adenoviral constructs for GFP (AdGFP), wild-type caveolin 1 (AdWTCav), or a caveolin 1 mutant lacking the scaffold domain (AdCavΔ) indicates significant overexpression of AdWTCav and AdCavΔ (A and B). There is significantly higher ERα (C and D) and caveolin 1 (C and E) in the plasma membrane in the AdWTCav-transduced cells. However, both ERα (C and D) and caveolin 1 (C and E) are significantly decreased in the AdCavΔ-transduced cells (C and E). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (C). Immunoprecipitation (IP) of caveolin 1 from whole-cell extracts followed by IB with ERα identified a significant increase in ERα bound to caveolin 1 in both AdWTCav- and AdCavΔ-transduced cells (F and G). Data are mean ± sem; n = 3. *, P < 0.05 vs. AdGFP-transduced cells.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Western Blot, Transduction, Construct, Mutagenesis, Over Expression, Marker, Immunoprecipitation

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Amino acids 62–73 of caveolin 1 span the ERα interaction region. Probing a peptide array representing the entire sequence of human caveolin 1 with recombinant human ERα (100 ng/ml) identified a single highly reactive peptide spanning amino acids 62–73 (shown by arrow in panel A). TAT-Cav62–73, along with a scrambled version containing a similar amino acid composition (TAT-Cav62–73scr), was synthesized as a fusion with the N-terminal TAT protein transduction domain from the HIV (B). PAEC were exposed to each peptide (100 ng/ml). Immunopreciptation (IP) analyses demonstrate that TAT-Cav62–73 significantly attenuates the association of ERα with caveolin 1 (C and D). Western blot analysis also demonstrates that TAT-Cav62–73 significantly attenuates ERα levels in the plasma membrane without altering total ERα levels (E and F; *, P < 0.05 vs. TAT-Cav62–73scr). Immunoblotting (IB) with the plasma membrane marker 5′-nucleotidase was also carried out to normalize for protein loading of the plasma membrane fractions (E). TAT-Cav62–73 and TAT-Cav62–73scr peptide-treated cells were transfected with an ERα-GFP construct and the plasma membrane labeled with Alexa Fluor 594 WGA (red). The extent of plasma membrane localization of ERα was determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of Alexa Fluor 594 WGA, and green fluorescence of ERα-GFP). TAT-Cav62–73 significantly attenuates the yellow fluorescence (G and H, the white bar in each panel represents 30 μm). TAT-Cav62–73 did not alter eNOS localization to the plasma membrane (I and J). n = 3; *, P < 0.05 vs. TAT-Cav62–73scr.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques: Peptide Microarray, Sequencing, Recombinant, Synthesized, Transduction, Western Blot, Marker, Transfection, Construct, Labeling, Fluorescence

Journal:

Article Title: Caveolin 1 Is Required for the Activation of Endothelial Nitric Oxide Synthase in Response to 17?-Estradiol

doi: 10.1210/me.2010-0043

Figure Lengend Snippet: Disrupting ERα-caveolin 1 interactions attenuates the interaction of ERα with PI3 kinase and inhibits downstream NO signaling. TAT-Cav62–73 significantly attenuates the 17β-estradiol-mediated association of ERα with PI3 kinase (A and B), the increase in eNOS phosphorylation at serine 1177 (C and D), and the increase in NO generation (E). TAT-Cav62–73 did inhibit the increase in NO generation in response to stimulation by shear stress (20 dyn/cm2, 15 min; panel F) or VEGF (100 ng/ml, 15min; panel G). Data are mean ± sem; n = 3–6. *, P < 0.05 vs. untreated; †, P < 0.05 vs. TAT-Cav62–73scr with 17β-estradiol.

Article Snippet: Generation and analysis of a caveolin 1 peptide array A library of overlapping peptides (12 mers), each shifted by five amino acids across the entire sequence of caveolin 1, was immobilized on cellulose membranes (Intavis Bioanalytical Instrruments AG, Cologne, Germany) and probed with 100 ng/ml recombinant ERα.

Techniques:

Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: Infrared gel electrophoresis images of DNase I footprinting products following incubation of compounds 1–6 with TOPOIIα promoter DNA. Compounds were incubated for 17 h with TOPOIIα promoter DNA IR-labelled on the sense (A) or antisense (B) strand. Each gel consists of 50 lanes: two GA marker lanes (GA) and six sets of eight lanes for compound (l–r; 0, 0.0016, 0.008, 0.04, 0.2, 1, 5 and 25 μM). The sequence positions relating to Supplementary Figure 1 are given vertically.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Nucleic Acid Electrophoresis, Footprinting, Incubation, Marker, Sequencing

Journal: Nucleic Acids Research

Article Title: A 96-well DNase I footprinting screen for drug–DNA interactions

doi: 10.1093/nar/gkm467

Figure Lengend Snippet: A comparison of footprint profiles of compounds 1–6 on the proximal region of the TOPOIIα promoter. Single footprint profiles were program-generated starting with the IR footprint gel image shown in A. Where no footprinting is seen, the sequence has been given an arbitrary score of −3.9 (log 125 μM). The DNA sequence is given in Supplementary Figure 1.

Article Snippet: The proximal 705 bp of the human Topoisomerase II alpha (TOPOIIα) promoter was amplified by PCR from human genomic DNA (Promega) using primers TIIA-F (CACCGCACACAGCCTAC) and TIIA-R (TGGTGACGGTCGTGAAG) (Supplementary Figure 1).

Techniques: Generated, Footprinting, Sequencing