Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 6. Ro protected hRMECs from AGEs-induced injury via the EPAC1/AMPK signaling pathway. A. Venn diagram drawn by the intersection of the genes that increased in the control group compared to the AGEs group and the genes that increased in the Ro treatment group compared to the AGEs group. B. Expression levels of Epac1 in control, model, and Ro-treated groups. C. The top 10 of KEGG passway Signaling Pathways. D, E. Representative immunoblots showed Epac1 and p- AMPK protein level in hRMECs. F, G. Images of Epac1 staining in hRMECs. The fluorescence intensity was quantified by Image J. Scale bar, 5um. The results are expressed as mean ± SD, #P < 0.05 and ##P < 0.01 vs. the control, **P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Control, Expressing, Protein-Protein interactions, Western Blot, Staining, Fluorescence

Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 8. Ro regulates Epac1/AMPK signaling pathway to ameliorate DR in diabetic mice. A. The mitochondria in mouse retinal microvascular endothelial cells were analysed by TEM. Red arrows indicate mitochondria. Scale bar, 1μm. B, C. Expression levels of Epac1 protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of Epac1 were captured by TissueFAXS Cytometry. Scale bars: 200 µm. D, E. Expression levels of p-AMPK protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of p-AMPK were captured by TissueFAXS Cytometry. Scale bars: 200 µm. The results are expressed as mean ± SD, #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the control, *P < 0.05, **P < 0.001 and ***P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Expressing, Marker, Fluorescence, Cytometry, Control

Journal: Pharmacological research

Article Title: Ginsenoside Ro prevents endothelial injury via promoting Epac1/AMPK- mediated mitochondria protection in early diabetic retinopathy.

doi: 10.1016/j.phrs.2024.107562

Figure Lengend Snippet: Fig. 9. Retina-specific deletion of Epac1 reverses the therapeutic effects of Ro. A, E. Retinal thickness was determined by OCT. Scale bar, 100um. B, F. Detection of changes in retinal blood vessels in mice by FFA. C, G. Representative images of H&E staining of retinal tissue from the mice. Scale bar, 50μm. D. The mitochondria in mouse retinal microvascular endothelial cells were analysed by TEM. Red arrows indicate mitochondria. Scale bar, 1μm. H, I. Expression levels of p- AMPK protein in mouse retinal cells, and DAPI marker nuclei. Representative fluorescence images of p-AMPK were captured by TissueFAXS Cytometry. Scale bars: 200 µm. The results are expressed as mean ± SD, #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. the control, *P < 0.05, **P < 0.001 and ***P < 0.001 vs. AGEs group.

Article Snippet: Then, the membrane was blocked using 5 % skim milk solution in TBST for 2 h at room temperature and incubated with corresponding primary antibodies overnight at 4°C: β-actin (ABclonal, AC038, 1:2000); EPAC1 (Proteintech, 12572–1- AP, 1:2000); p-AMPK (Abcam, AB133448, 1:2000); AMPK (Abcam, AB32047, 1:2000); Bcl-2 (ABclonal, A0208, 1:2000); CD31/PECAM1 (ABclonal, A0378, 1:1000); p-DRP-1 (ABclonal, AP1353, 1:1000); DRP1 (ABclonal, A21968, 1:1000); PINK1 (ABclonal, A25301, 1:2000); Parkin (ABclonal, A0968, 1:1000)LC3 (Sigma-Aldrich, L8918, 1:2000); SQSTM1/p62 (ABclonal, 1:2000); ATG5 (ABclonal, A0203, 1:1000); ATG12 (ABclonal, A22788, 1:1000); Beclin-1 (ABclonal, A7353, 1:2000).

Techniques: Staining, Expressing, Marker, Fluorescence, Cytometry, Control

Journal: Journal of ethnopharmacology

Article Title: Anemarrhena asphodeloides Bunge and Phellodendri Chinensis Cortex inhibits the PTGS2/EP2/cAMP/Epac1 signaling pathway to reduce microglial M1 polarization, thereby blocking chronic stress-induced depression-like behavior.

doi: 10.1016/j.jep.2025.119792

Figure Lengend Snippet: Fig. 8. Neomangiferin alleviates the release of proinflammatory factors in BV2 cells by inhibiting PTGS2/EP2/cAMP/Epac1 signals. (A) representative immunofluorescence staining of EP2 and Epac1, and (B) fluorescence area (n = 3). (C–D) Protein expression changes of EP2 and Epac1 (n = 3). The contents of PGE2 and cAMP in BV2 cells were determined by E-F Elisa kit (n = 3). (G) Representative images of zebrafish embryos in Neomangiferin experiment (n = 10). Data are expressed as mean±SD. *** means P < 0.001,** means P < 0.01,* means P < 0.05, ns means P > 0.05.

Article Snippet: Seal with 5 % skim milk powder at room temperature for 2 h. Primary antibody β-actin (rabbit, 1:5000; bs-00R,Bioss, China), GAPDH(rabbit, 1:10,000; 10494- 1-AP,Proteintech, China), HSP90(rabbit, 1:5000; 13171-1-AP,Proteintech, China), Arg-1 (rabbit, 1:1000; 16001-AP, Proteintech, China), iNOS (rabbit, 1:1000; ab283655, Abcam, UK), PTGS2 (mouse, 1:400; 66351-1-Ig, Proteintech, China), EP2 (rabbit, 1:1000; HA721380, HUABIO, China), EPAC1 (rabbit, 1:1000; A02483-3, Boster, China), incubated overnight at 4 ◦C.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Cilostazol Induces PGI 2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

doi: 10.1371/journal.pone.0132835

Figure Lengend Snippet: (A) Top, Representative immunoblot showing PDE3A and PDE3B expression in human adipose, artery, and HAECs. Bottom, Fluorescence microscopy analysis of PDE3A and PDE3B expression in HAECs (green, VE-Cadherin; red, PDE3A or 3B). (B) Top, Effect of PDE3-targeting siRNAs (iPDE3A and 3B) or non-targeting siRNAs (iCt) on protein levels in HAEC. Bottom, Down-regulation of PDE3A and PDE3B expression increases intracellular cAMP levels in HAECs. HAECs were transfected with a iCT, iPDE3A, or iPDE3B. Post-transfection HAECs were treated with vehicle (Veh) or 30 μM cilostazol (C30), and then the intracellular cAMP concentration was determined. (C) Down-regulation of PDE3A and PDE3B expression increases PGI 2 production in HAECs. HAECs were transfected with a iCT, iPDE3A, or iPDE3B. Post-transfection HAECs were treated with vehicle (Veh) or 30 μM cilostazol (C30), and then the PGI 2 concentration in medium was determined (n = 4; †† p < 0.01 vs. iCT, t -test; ** p < 0.01 vs. Veh, t -test).

Article Snippet: The secondary antibodies, Alexa fluor488- and 568-conjugated antibodies, were from Life Technologies, Inc. (Carlsbad, CA, USA). siRNAs against PDE3A and PDE3B (Hs.591150 and Hs.445711, respectively), and control siRNA were purchased from Life Technologies, Inc. siRNA against Epac-1 (sc-41700), Rap-1 (sc-38554), and PLCε (sc-44024) were purchased from Santa Cruz Biotechnology, Inc. For Biacore analysis, human recombinant PI3Kγ protein was from OriGene Technologies, Inc. Biotin-labeled Epac-1-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) and biotin-labeled PI3Kγ-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) were purchased from Toray Research Center, Inc. (Tokyo, Japan).

Techniques: Western Blot, Expressing, Fluorescence, Microscopy, Transfection, Concentration Assay

Journal: PLoS ONE

Article Title: Cilostazol Induces PGI 2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

doi: 10.1371/journal.pone.0132835

Figure Lengend Snippet: (A) Effect of cAMP effectors on PGI 2 production. HAECs were treated with vehicle (0.05% DMSO), cilostazol (30 μM), 007 (10 μM), or 6-Bn-cAMP (n = 4; †† p < 0.01 vs. vehicle, ** p < 0.01 vs. cilostazol, t -test). (B) Top, Representative immunoblot showing total PKA type IIβ (RIIβ) expression in HAECs. Bottom, Effect of the PKA inhibitor, 14–22 amide, on PGI 2 production in HAECs (n = 4; †† p < 0.01 vs. vehicle, t -test). (C) Top, Representative immunoblot showing total Epac-1 expression in HAECs. Middle, Effect of Epac-1-targeting siRNAs (iEpac-1) or non-targeting siRNAs (iCt) on protein levels in HAEC. Bottom, Effect of Epac-1 knockdown on PGI 2 production in HAECs. HAECs were transfected with iEPAC-1, or with iCT. Post-transfection HAECs were treated with vehicle or 30 μM cilostazol (n = 4; † p < 0.05 vs. vehicle t -test).

Article Snippet: The secondary antibodies, Alexa fluor488- and 568-conjugated antibodies, were from Life Technologies, Inc. (Carlsbad, CA, USA). siRNAs against PDE3A and PDE3B (Hs.591150 and Hs.445711, respectively), and control siRNA were purchased from Life Technologies, Inc. siRNA against Epac-1 (sc-41700), Rap-1 (sc-38554), and PLCε (sc-44024) were purchased from Santa Cruz Biotechnology, Inc. For Biacore analysis, human recombinant PI3Kγ protein was from OriGene Technologies, Inc. Biotin-labeled Epac-1-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) and biotin-labeled PI3Kγ-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) were purchased from Toray Research Center, Inc. (Tokyo, Japan).

Techniques: Western Blot, Expressing, Transfection

Journal: PLoS ONE

Article Title: Cilostazol Induces PGI 2 Production via Activation of the Downstream Epac-1/Rap1 Signaling Cascade to Increase Intracellular Calcium by PLCε and to Activate p44/42 MAPK in Human Aortic Endothelial Cells

doi: 10.1371/journal.pone.0132835

Figure Lengend Snippet: (A) Cilostazol, cilostamide, and milrinone interfere with association of PDE3B-binding Epac-1 peptides-1 to PDE3B. The above-mentioned compounds’ competition with PDE3B-binding Epac-1 peptides-1 (5 μM) binding to Epac-1-binding PDE3B peptide was evaluated (n = 4; ** p < 0.01 vs. 5 μM PDE3B-binding Epac-1 peptides-1, randomized Dunnett’s test). (B) Cilostazol, cilostamide, and milrinone interfere with association of PDE3B-binding Epac-1 peptides-2 to PDE3B. These compounds’ competition with Epac-1 peptides-2 (5 μM) binding to Epac-1-binding PDE3B peptide was evaluated (n = 4; * p < 0.05, ** p < 0.01 vs. 5 μM PDE3B-binding Epac-1 peptides-2, randomized Dunnett’s test). (C) Direct bindings of cilostazol, cilostamide, and milrinone to PI3Kγ-binding PDE3B peptide. Relative responses of PI3Kγ-binding PDE3B peptide to drugs at concentrations of 0.3125, 0.625, 1.25, 2.5, and 5 μM (n = 4).

Article Snippet: The secondary antibodies, Alexa fluor488- and 568-conjugated antibodies, were from Life Technologies, Inc. (Carlsbad, CA, USA). siRNAs against PDE3A and PDE3B (Hs.591150 and Hs.445711, respectively), and control siRNA were purchased from Life Technologies, Inc. siRNA against Epac-1 (sc-41700), Rap-1 (sc-38554), and PLCε (sc-44024) were purchased from Santa Cruz Biotechnology, Inc. For Biacore analysis, human recombinant PI3Kγ protein was from OriGene Technologies, Inc. Biotin-labeled Epac-1-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) and biotin-labeled PI3Kγ-binding PDE3B peptide (Met-1 to Glu-25; MRRDERDAKAMRSLQPPDGAGSPPE-K-biotin-NH2) were purchased from Toray Research Center, Inc. (Tokyo, Japan).

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

doi: 10.1038/srep36370

Figure Lengend Snippet: (A) Lysates from HEK293 cells overexpressing YFP-Epac1 or GFP were subjected to GFP-TRAP immunoprecipitation (IP) followed by silver staining and mass spectrometry (MS) analyses of bands between 72 and 120 kDa. Mass spectrometry results are presented in . ( B ) GFP-TRAP precipitates from HEK293 cells expressing YFP-Epac1, YFP-Epac2, or GFP as a negative control, were analyzed by western blotting with impβ1 and GFP antibodies. Levels of impβ1, Epac1, and Epac2 in the IP and total lysate (TL) are shown in representative western blots. (C) Impβ1 was immunoprecipitated from cells expressing HA-Epac1 or control vector using impβ1 antibody coupled to protein G-sepharose. Samples were analyzed by western blotting with impβ1 and Epac1 antibodies. Representative western blots are shown. (D) Similar to (B), GFP-TRAP precipitates from N2A cells expressing YFP-Epac1, YFP-Epac2, or GFP as a negative control, were analyzed by western blotting. (E) Endogenous Epac1 was immunoprecipitated from EA.hy926 cells using Epac1 antibody (A-5) coupled to protein A-sepharose. Proteins were visualized by western blotting using Epac1 and impβ1 antibodies.

Article Snippet: Flow FRET assays of cells transfected with the FRET probe ECFP-Epac1-citrine were performed using an LSR Fortessa X-20 Analyzer (BD Biosciences).

Techniques: Immunoprecipitation, Silver Staining, Mass Spectrometry, Expressing, Negative Control, Western Blot, Plasmid Preparation

Journal: Scientific Reports

Article Title: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

doi: 10.1038/srep36370

Figure Lengend Snippet: (A) N2A cells co-expressing YFP-Epac1 (green) and mcherry-impβ1 (red) were treated with either vehicle or 1 μM 8-pCPT-AM for 15 min, followed by live cell imaging. Representative images of >50 cells in each condition are shown; scale bar corresponds to 25 μm. (B) N2A cells expressing YFP-Epac1 or GFP were treated with either vehicle or 10 μM 8-pCPT-AM for 15 min. Epac1 was pulled down using the GFP-TRAP beads and samples were analyzed by western blotting with impβ1 and Epac1 antibodies. (C) N2A cells overexpressing YFP-Epac1 were treated with impβ1 si-RNA (si-impβ1) or control si-RNA (si-ctl) and the effect on Epac1 localization was monitored by live cell imaging. Images are representative of at least three independent experiments with >50 cells each; scale bar corresponds to 25 μm. Western blot shows confirmation of impβ1 knockdown. (D) PM sheets of control (ctl) EA.hy926 cells or EA.hy926 cells treated with 1 μM 8-pCPT-AM for 15 min (left panels), or EA.hy926 cells treated with si-impβ1 or si-ctl (right panels) were attached to EM grids, immunolabeled with gold nanoparticles coupled to Epac1 antibody (H-70), and imaged by EM. Images are representative of at least 15 PM sheets per condition; scale bar corresponds to 200 nm. Quantification of the number of gold-coupled Epac1 nanoparticles per μm 2 area in the inner leaflet of the plasma membrane is shown in the bar graphs. Data were analyzed by Student’s t-test; ***P < 0.001 .

Article Snippet: Flow FRET assays of cells transfected with the FRET probe ECFP-Epac1-citrine were performed using an LSR Fortessa X-20 Analyzer (BD Biosciences).

Techniques: Expressing, Live Cell Imaging, Western Blot, Immunolabeling

Journal: Scientific Reports

Article Title: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

doi: 10.1038/srep36370

Figure Lengend Snippet: (A) N2A cells overexpressing YFP-Epac1 or GFP, were cultured in serum-free medium (SFM; solid bars) to induce differentiation or kept in regular growth medium (RM; open bars). Cells were also treated with impβ1 si-RNA (si-impβ1) or control si-RNA (si-ctl). Neurite length was analyzed after 24 hrs as described in the methods section. Data shown are the mean ± SEM neurite lengths for three independent experiments where at least 4 images of >40 cells each were analyzed for each condition. Data were analyzed by Two-Way ANOVA; ***P < 0.0001 . (B) Representative images of cells from each condition cultured in serum free medium; YFP-Epac1 (green), βIII-tubulin (red), and DAPI (blue); scale bar corresponds to 25 μm.

Article Snippet: Flow FRET assays of cells transfected with the FRET probe ECFP-Epac1-citrine were performed using an LSR Fortessa X-20 Analyzer (BD Biosciences).

Techniques: Cell Culture

Journal: Scientific Reports

Article Title: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

doi: 10.1038/srep36370

Figure Lengend Snippet: (A) Representative images of N2A cells expressing Epac1-CAAX to tether Epac1 to the PM or control Epac1; scale bar corresponds to 25 μm. (B) N2A cells expressing Epac1 or Epac1-CAAX were grown in either SFM (solid bars) or RM (open bars) for 24 hrs. Data shown are the mean ± SEM neurite lengths for three independent experiments where at least 4 images of >40 cells each were analyzed for each condition. Comparison between Epac1 (SFM) and Epac1-CAAX (SFM) was performed by Student’s t-test; ***P < 0.0001 . (C) Cells expressing YFP-Epac1-R82A mutant (deficient in binding phosphatidic acid at the PM) and treated with si-ctl or si-impβ1, were analyzed by live cell imaging. Representative images show that YFP-Epac1-R82A does not localize to the PM when impβ1 is knocked down; scale bar corresponds to 25 μm. Confirmation of the inability of Epac1-R82A to accumulate at the PM in response to 1 μM 8-pCPT-AM is shown in the lower left image. Western blot confirms impβ1 knockdown. (D) Cells expressing either YFP-Epac1 or YFP-Epac1-R82A mutant and treated with either si-ctl or si-impβ1 were analyzed for neurite outgrowth after culture in serum-free medium for 24 hrs. Data analyzes were performed by Two-Way ANOVA; ***P < 0.0001 .

Article Snippet: Flow FRET assays of cells transfected with the FRET probe ECFP-Epac1-citrine were performed using an LSR Fortessa X-20 Analyzer (BD Biosciences).

Techniques: Expressing, Mutagenesis, Binding Assay, Live Cell Imaging, Western Blot

Journal: Scientific Reports

Article Title: Epac1 interacts with importin β1 and controls neurite outgrowth independently of cAMP and Rap1

doi: 10.1038/srep36370

Figure Lengend Snippet: (A) N2A cells overexpressing the Epac1 FRET reporter were treated with si-ctl or si-impβ1 and analyzed by flow cytometry following treatment with either vehicle or 30 μM 8-pCPT-AM for 15 min. Bar graph shows the percentage of the FRET ratio (405/530) in YFP-positive cells, in si-ctl (open bars) as compared to si-impβ1 (solid bars) samples. (B) N2A cells overexpressing YFP-Epac1 were treated with si-ctl or si-impβ1, followed by treatment with vehicle or 0.1 μM 8-pCPT-AM for 15 min. Rap1-GTP was pulled down with Ral GDS-Rap binding domain beads followed by western blotting to detect Rap1-GTP and total Rap1, impβ1, and Epac1. (C) Binding of purified Epac1 to membrane lipid strips in the absence or presence of 50 μM 8-pCPT for 15 min. (D) Representative images of N2A cells overexpressing YFP-Epac1-R279L mutant (deficient in binding cAMP) and treated with si-ctl or si-impβ1 were analyzed by live cell imaging. Representative images show that YFP-Epac1-R279L localizes to the PM when impβ1 is knocked down. Lower left image confirms that Epac1-R279L does not respond to stimulation with 8-pCPT-AM. (E) N2A cells overexpressing either YFP-Epac1-R279L mutant or YFP-Epac1 and treated with si-ctl or si-impβ1, were grown in SFM (solid bars) or RM (open bars). Neurite length was analyzed as described in the legend to . ( F ) The Epac inhibitor ESI-09 does not prevent the effect of impβ1 depletion on PM accumulation of Epac1. N2A cells overexpressing YFP-Epac1 were treated with si-ctl or si-impβ1 and 5 μM ESI-09. Representative images showing that YFP-Epac1 also localizes to the PM in the presence of ESI-09 when impβ1 is knocked down. (G) N2A cells overexpressing YFP-Epac1 treated with si-ctl or si-impβ1, were grown in SFM supplemented with vehicle (open bars) or 5 μM ESI-09 (solid bars) for 24 hrs. Data in all panels were analyzed by Two-Way ANOVA; ns: not significant, ***P < 0.001 , and **P < 0.01 . Scale bar in all images corresponds to 25 μm.

Article Snippet: Flow FRET assays of cells transfected with the FRET probe ECFP-Epac1-citrine were performed using an LSR Fortessa X-20 Analyzer (BD Biosciences).

Techniques: Flow Cytometry, Binding Assay, Western Blot, Purification, Mutagenesis, Live Cell Imaging

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: The N-terminal 49 amino acids of Epac1 contribute to Epac1-mediated cell adhesion. (A) Domain architecture of Epac1. Epac1 contains a CDC25 homology domain (CDC25-HD), which mediates GEF activity, a Ras exchange motif (REM), which stabilizes the CDC25-HD, a cyclic nucleotide binding (CNB) domain, a DEP (Dishevelled, Egl-10, and pleckstrin) domain, and a Ras association (RA) domain. (B) Adhesion of Jurkat T cells transfected with empty vector (EV), wild-type (Wt) Epac1, or different Epac1 mutants together with a luciferase reporter. Transfected cells were allowed to adhere to a fibronectin-coated surface for 45 min, and adhesion was subsequently detected by measurement of luciferase activity. The induction of adhesion by activation of Epac1 with 007 (100 μM) is partially impaired with the Epac1 mutants lacking the DEP domain (Epac1ΔDEP) or N49 (Epac1Δ1-49), whereas adhesion is completely abrogated when both the DEP domain and N49 are deleted (Epac1Δ1-148). Shown are average data with standard deviations from three individual experiments, with adhesion normalized to 100% for 007-activated wild-type Epac1. Statistical analysis was performed using a one-tailed Student t test. The Western blot labeled with the Epac1 antibody (5D3) shows expression levels of the transfected Epac1 constructs. (C) Jurkat T cells were transfected with similar amounts of either wild-type Epac1, Epac1ΔDEP, Epac1Δ1-49, or Epac1Δ1-148, and GTP-bound Rap1 was pulled down from lysates of cells after stimulation with 007 (100 μM, 10 min).

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques: Activity Assay, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activation Assay, One-tailed Test, Western Blot, Labeling, Expressing, Construct

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: Epac1 directly interacts with ERM proteins. (A) HA-Epac1 coimmunoprecipitation (IP) with Flag-ezrin, Flag-radixin, and Flag-moesin in HEK293 cells. TL, total lysate. (B) Coimmunoprecipitation of Flag-radixin with YFP-tagged wild-type Epac1, Epac1Δ1-49, and Epac1ΔDEP in HEK293 cells. The 49 N-terminal amino acids of Epac1 are required for the interaction with radixin. (C) Coimmunoprecipitation of Flag-radixin with HA-tagged versions of Epac1 or Epac2 in HEK293 cells. In contrast to HA-Epac1, HA-Epac2 is unable to coimmunoprecipitate Flag-radixin.

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: ERM proteins require the open conformation to bind Epac1. (A) Domain architecture of radixin. The FERM (4.1 protein, ezrin, radixin, moesin) domain and actin binding domain (ABD) form an intramolecular interaction and are linked by an α-helical region. Phosphorylation of T564 is required for the open conformation of radixin, and I577 and I580 are part of the hydrophobic interaction surface between the ABD and the FERM domain. (B) Coimmunoprecipitation of HA-Epac1 with Flag-tagged wild-type radixin and radixin lacking the ABD (Flag-radixinΔABD, residues 1 to 492) in HEK293 cells. The separate N terminus of radixin shows enhanced binding to Epac1 compared to full-length radixin, implying that the presence of the C-terminal ABD suppresses binding of radixin to Epac1. (C) Coimmunoprecipitation of HA-Epac1 with wild-type Flag-radixin and the constitutively open mutants Flag-radixin(T564D) and Flag-radixin(I577D, F580D) in HEK293 cells. The amount of coimmunoprecipitated HA-Epac1 is significantly increased with these radixin mutants. (D) Live imaging of YFP-tagged wild-type radixin and the constitutively open mutant radixin(T564D) in HEK293 cells, showing that upon conformational opening radixin is redistributed to the PM. (E) Live imaging of Epac1 in HEK293 cells. The indicated constructs were expressed alone or with Flag-radixin(T5645D. (F) 007-AM (1 μM) treatment of HEK293 cells transfected with the FRET sensor CFP-Epac1ΔDEP-YFP. The sensor reports the cAMP-induced conformational change as a loss of intramolecular FRET; deletion of the DEP domain precludes changes in intermolecular FRET due to cAMP-dependent relocalization (51). When this Epac1 FRET construct is recruited to the PM by coexpressed radixin(T564D), its conformational opening upon 007-AM treatment remains unaffected. Scale bars (all images), 10 μm.

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques: Binding Assay, Imaging, Mutagenesis, Construct, Transfection

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: GPCR-mediated activation of ERM proteins induces binding of Epac1. (A) Coimmunoprecipitation of HA-Epac1 with Flag-radixin in HEK293 cells stimulated with thrombin (0.2 U/ml, 2 min). Thrombin stimulation results in increased phosphorylation of the ERM proteins (ezrin T567, radixin T564, and moesin T558) (right) and the enhanced interaction of HA-Epac1 with Flag-radixin. (B) Quantification of the relative binding of Epac1 to radixin from three independent experiments, as performed for panel A. Statistical analysis was performed using a one-tailed Student t test. (C) Measurement of FRET between YFP-radixin and Epac1-TdTom or Epac1Δ1-49-TdTom in HEK293 cells during stimulation with thrombin receptor-activating peptide (TRP; 50 μM). FRET between YFP-radixin and Epac1-TdTom, expressed as ratio of TdTom/YFP fluorescence, increases upon TRP addition, confirming that binding to Epac1 is increased when radixin is driven in the open conformation. The traces are representative of three independent measurements. The average increase (±standard deviation) in the ΔTdTom/YFP ratio was 6.0% ± 1.1%. (D) Coimmunoprecipitation of endogenous ezrin with endogenous Epac1 in ACHN cells with or without prestimulation with sphingosine 1-phosphate (S1P; 1 μM) to induce the conformational opening of ezrin.

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques: Activation Assay, Binding Assay, One-tailed Test, Fluorescence, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: GPCR-mediated activation of ERM proteins induces a clustered localization of Epac1 at the PM. (A) Live imaging of Epac1-YFP before and after 50 μM TRP stimulation to induce phosphorylation and conformational opening of ERM proteins in HEK293 cells. In response to TRP, Epac1-YFP has a clustered localization at the PM. Quantitative analysis reveals the rapid kinetics of TRP-induced recruitment of Epac1 in this experiment. Shown are fluorescence intensities at the right flank of the plasma membrane (PM; red) and in the cytosol (blue) as well as the PM/cytosol ratio (green). (B) Live imaging of Epac1Δ1-49-YFP before and after 50 μM TRP stimulation in HEK293 cells. Epac1Δ1-49-YFP is not targeted to the PM in response to TRP. (C) Live imaging of YFP-radixin and Epac1-TdTom after 50 μM TRP stimulation to induce phosphorylation and conformational opening of radixin in HEK293 cells. In response to TRP, both constructs are similarly clustered at the PM. (D) Subcellular localization of Epac1-YFP and endogenous radixin after 50 μM TRP stimulation in HEK293 cells. (E) Live imaging of HEK293 cells that were transfected with CFP-N49 and Epac1Δ1-49-YFP and stimulated with TRP (50 μM) and 007-AM (1 μM). Both constructs accumulate at the PM but show different subcellular distributions. (F) Live imaging of Epac1-YFP in HEK293 cells, showing the inhibition of plasma membrane recruitment by TRP (50 μM) by preincubation with the exoenzyme C3, an inhibitor of RhoA (16 h, 30 μg/ml). (G) Live imaging of Epac1-YFP in HEK293 cells, showing the recruitment of Epac1 to the plasma membrane by coexpression of the catalytic domain of p190-RhoGEF. The clustered localization of Epac1 remains upon activation of Epac1 by stimulation with 007-AM (1 μM). Scale bars (all images), 10 μm.

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques: Activation Assay, Imaging, Fluorescence, Construct, Transfection, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Spatial Regulation of Cyclic AMP-Epac1 Signaling in Cell Adhesion by ERM Proteins ▿ ¶

doi: 10.1128/MCB.00463-10

Figure Lengend Snippet: ERM binding is required for efficient Epac1-mediated cell adhesion. (A) Coimmunoprecipitation of HA-Epac1 with Flag-radixin in the presence of the V5-tagged C-terminal ABD of radixin in HEK293 cells. Overexpression of V5-ABD decreases the interaction of HA-Epac1 with Flag-radixin. (B) Live imaging of YFP-Epac1 in HEK293 cells coexpressed with CFP-radixin ABD. In the presence of CFP-radixin ABD, plasma membrane recruitment by TRP (50 μM) is inhibited, whereas the subsequent 007-AM (1 μM)-induced translocation of Epac1 remained unaffected. (C) Adhesion of Jurkat T cells that stably express Epac1 transiently transfected with either empty vector (EV) or the ABD of radixin to compete for binding of Epac1 to endogenous ERM proteins. Transfected cells were allowed to adhere to a fibronectin-coated surface for 45 min, and adhesion was subsequently detected by measuring the activity of cotransfected luciferase. Adhesion induced by activation of Epac1 with 007 (100 μM) is decreased by overexpression of the ABD of radixin. Shown are average data with standard deviations from three individual experiments, with 007-induced adhesion in EV-transfected cells normalized to 100%. Statistical analysis was performed using a one-tailed Student t test. The Western blot labeled with the V5 and Epac1 antibody (5D3) shows the expression of Epac1 and the transfected V5-radixin ABD construct. (D) Adhesion of Jurkat T cells transiently transfected with YFP-Δ1-49-Epac1 and either EV or the ABD of radixin. Adhesion of transfected cells was measured by a method similar to the method used for Fig. ​Fig.5C.5C. Results show that the radixin ABD does not reduce adhesion induced by YFP-Δ1-49-Epac1. Shown are average data with standard deviations from three individual experiments, with 007-induced adhesion in EV-transfected cells normalized to 100%. Statistical analysis was performed using a one-tailed Student t test. The Western blot labeled with the V5 and Epac1 antibody (5D3) shows the expression of YFP-Δ1-49-Epac1 and the V5-radixin ABD construct. (E) Adhesion of Ovcar3 cells transfected with either control or ezrin, radixin, and moesin SMARTpool siRNAs. Sixty hours after siRNA transfection, cells were allowed to adhere to a fibronectin-coated surface for 45 min in the absence or presence of 100 μM 007, and adhesion was subsequently detected by measuring endogenous phosphatase activity. Shown are average data with standard deviations from two individual experiments, normalized to adhesion of control siRNA-transfected cells. Statistical analysis was performed using a one-tailed Student t test. The Western blot shows the expression of endogenous ezrin, radixin, moesin, and tubulin (as a loading control).

Article Snippet: Human full-length Epac1 (RapGEF3; Homo sapiens ; GI 3978530), cloned using a pB27 vector, was screened with a randomly primed human placenta library by Hybrigenics S.A. (Paris, France), as previously described ( 54 ).

Techniques: Binding Assay, Over Expression, Imaging, Translocation Assay, Stable Transfection, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Activation Assay, One-tailed Test, Western Blot, Labeling, Expressing, Construct