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: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: TRPC3 expression and SOCE in melanoma. a Representative images of immunohistochemical staining of HE, MART1, and TRPC3 in a melanoma primary tissue microarray (stage II) (original magnification, b ×200). The calibration bars represent 200 µm. b mRNA expression in various melanoma cell lines. SK-Mel-2 is a skin metastasis melanoma cell line with NRAS mutation, SK-Mel-24 is a lymph node metastasis cell line with BRAFV600E mutation, SK-Mel-187 is also a lymph node metastasis cell line, C8161 is a metastasis cell line with wild-type BRAF, and HEMA-LP is a skin melanocyte cell line. TRPC3 mRNA is widely expressed in human melanoma cell lines, independently of BRAF mutation. c Immunoprecipitation for TRPC3 and STIM1 was performed. d Cytosolic Ca2+ level in C8161 cells is shown as mean ± SD (n = 8–9). SOCE was examined in the presence or absence of DMSO (vehicle control 1 µM) or Pyr3 (1 µM) in C8161. Pyr3 inhibited SOCE in C8161 cells. Pyr3 was added after the addition of Ca2+. The Ca2+ signal was immediately decreased, indicating that Pyr3 inhibits SOCE in melanoma cells. Data in each panel are averages of eight or nine cells

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray, Mutagenesis, Immunoprecipitation, Control

Journal: The Journal of Physiological Sciences : JPS

Article Title: Transient receptor potential cation 3 channel regulates melanoma proliferation and migration

doi: 10.1007/s12576-016-0480-1

Figure Lengend Snippet: Pyr3 inhibits phosphorylation of STAT5 and Akt. a Representative images of Akt phosphorylation are shown. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of Akt was inhibited by TRPC3 inhibitor, Pyr3 (n = 4, **p < 0.01, ns not significant). b Protein phosphorylation microarray analysis in the presence of Pyr3. C8161 cells were incubated with DMSO (vehicle control) or Pyr3 (10 μM) for 15 min. The Y-axis shows the signal ratio of phosphorylated to non-phosphorylated protein in the presence of Pyr3 as a percentage of that of the DMSO control. c Representative images of STAT5 phosphorylation. Densitometric analyses (bar graph) of Western blots showed that phosphorylation of STAT5 was inhibited by Pyr3 (n = 4, *p < 0.05, ns not significant)

Article Snippet: C8161 cells treated with DMSO or Pyr3 (10 μM) for 15 min was subjected to protein phosphorylation microarray assay using a commercial kit (Cancer Signaling Phospho-Antibody Array; Full Moon BioSystems, Inc.).

Techniques: Phospho-proteomics, Western Blot, Microarray, Incubation, Control

Journal: bioRxiv

Article Title: Cardiac fibroblasts regulate cardiomyocyte hypertrophy through dynamic regulation of type I collagen

doi: 10.1101/2022.05.25.493406

Figure Lengend Snippet: (A) Whole ventricle mRNA microarray analysis of Col1a2 -/- mouse hearts compared to Col1a2 +/- at 2 months of age, n=3 per genotype. (B) Mass spectrometry analysis of ECM protein changes in Col1a2 -/- mouse hearts compared to Col1a2 +/- hearts at 3 months of age, n=4 per genotype. (C) Representative immunofluorescence images and (D) Western blot analysis of periostin from hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Scale bar: 25 µm. (E) Flow cytometric gate strategy and (F) analysis of cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from dissociated hearts of Col1a2 +/- and Col1a2 -/- mice at 3 months of age. (G) Representative immunofluorescence images of platelet-derived growth factor receptor (PDGFR)-α (purple) in Col1a2 +/- and Col1a2 -/- mice at 3 months of age. Wheat germ agglutinin (WGA) staining is green and shows outlines of cardiomyocytes. Scale bar: 100 µm. Relative mRNA expression of Col1a2 (H), Postn (I), Col3a1 (J) and Col5a1 (K) in sorted cardiac fibroblasts (MEFSK4 + /CD31 - /CD45 - ) from Col1a2 +/- and Col1a2 -/- mice at 9 months of age. Student t -test for panels (F), (H), (I), (J) and (K).

Article Snippet: Antibodies against the following proteins were used: periostin (Novus Biologicals NBP1-30042; 1:300 dilution for IF, 1:1000 for Western blot); collagen I (Abcam ab21286; 1:100 for IF); PDGFRα from (R&D Systems AF1062; 1:1000 for IF); collagen 1a2 (Santa Cruz sc-393573; 1:500 for Western blot) Anti-CD31 was from BioLegend (102423; 1:100 for flow cytometry); anti-CD45 was from BD Biosciences (563890; 1:100 for flow cytometry); anti-MEFSK4 was from Miltenyi Biotec (130-120-802; used 1:30 for flow cytometry).

Techniques: Microarray, Mass Spectrometry, Immunofluorescence, Western Blot, Derivative Assay, Staining, Expressing

Journal: Nature Communications

Article Title: Primary cilia and SHH signaling impairments in human and mouse models of Parkinson’s disease

doi: 10.1038/s41467-022-32229-9

Figure Lengend Snippet: a Immunostainings exemplarily shown for O3H-R1-003. hiPSC pluripotency staining for markers OCT4, NANOG, SOX2. Scale bar=200 µm. hNPC staining for markers SOX1, SOX2, NESTIN, PAX6. Scale bar=100 µm. Neuron staining for markers TUBB3 and DAn marker TH. Scale bar=100 µm. Astrocyte staining for markers GFAP and SLC1A3. Scale bar=100 µm. b Summary of somatic CNVs identified in hNPC clones by chromosomal microarray analysis shown as total number of somatic CNVs detected per analyzed clone and average length of CNVs (in kb; green=copy number gain; orange=copy number loss) per analyzed clone. n = 5 Ctrl and 7 sPD patients. c Circos plot showing the genomic distribution of somatic CNVs in Ctrl (blue) and sPD (red) clones. d Quantification of RBFOX3 (synonym: NeuN) positive as well as TH / RBFOX3 double-positive cells in DAn populations. n = 5 Ctrl and 7 sPD clones, in triplicates. e Characterization of neurite morphologies of DAns. Boxplots show the average number of neurites emerging from TH positive cell bodies, their average number of branch points and their average length. n = 5 Ctrl and 7 sPD clones, in triplicates. Boxplots display the median and range from the 25th to 75th percentile. Whiskers extend from the min to max value. Each dot represents one patient. P -values were determined by two-sided t -test d (right), e ; two-sided Mann–Whitney-U test b, d (left). * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies were diluted as follows: AC-TUB (T6793, Sigma-Aldrich; 1:1000), ARL13B (17711-1-AP, Proteintech; 1:500), GFAP (MAB360, Millipore; 1:250), GLI3 (AF3690, R&D; 1:100), NANOG (AF1997, R&D Systems; 1:200), NES (Ma1110, Thermo Fisher Scientific; 1:250), PAX6 (Ab78545, Abcam; 1:200), PITX3 (38-2850, Invitrogen; 1:300), POU5F1 (2840 S, Cell Signaling; 1:500), RBFOX3 (ab104224, Abcam; 1:800), SLC1A3 (NB100-1869, Novus Biologicals; 1:250), SMO (sc166685, Santa Cruz; 1:500), SOX1 (Ab87775, Abcam; 1:500), SOX2 (sc17320, Santa Cruz; 1:500), TH (P40101, PelFreez; 1:600), TUBB3 (T5076, Sigma-Aldrich; 1:1000).

Techniques: Staining, Marker, Clone Assay, Microarray, Full Display Name, MANN-WHITNEY