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: The Journal of Neuroscience

Article Title: Application of an Epac Activator Enhances Neurotransmitter Release at Excitatory Central Synapses

doi: 10.1523/JNEUROSCI.0268-08.2008

Figure Lengend Snippet: Expression of Epac in neuronal cell cultures. A, Cultured autaptic neurons express both Epac isoforms. Lanes, Rat cerebellum lysate, positive control (+), murine dentate gyrus neurons (m-DG), murine CA1 neurons (m-CA1), rat dentate gyrus neurons (r-DG), rat CA1 neurons (r-CA1). Protein bands were detected at the following molecular weights: 126 kDa for Epac2, 100 kDa for Epac1, and 43 kDa for β-actin. Epac1 experiments were performed in triplicate. B1, Test for the specificity of the Epac-activating cAMP analog, ESCA1. A Western blot analysis of cAMP- or cAMP-analog-mediated phosphorylation of synapsin I at its PKA phosphorylation site (Ser9) is shown. For drug treatment procedure, see Materials and Methods. β-Actin controls for equal protein loading. B2, Quantitative change in the phosphorylation level of the PKA site of synapsin I. To correct for nonspecific effects (unrelated to drug treatment), which might have been introduced by variability in protein loading, P-Ser9 synapsin I chemiluminescence intensities were first normalized to the intensities of the corresponding β-actin values. These values were further normalized to the control intensity value to determine the increase or decrease in P-Ser9 levels after drug treatment. Values are shown as mean ± SEM based on the number of different Western blot performances. Statistical significance was tested by an unpaired two-tailed Student's t test assuming unequal variances (see Materials and Methods). Numbers within histogram bars represent the number of measurements.

Article Snippet: The antibodies (polyclonal) were applied in the following dilutions: phospho-synapsin (Ser9; Cell Signaling Technology), 1:1000; β-actin (I-19; Santa Cruz Biotechnology), 1:500; Epac1 (GeneTex), 1:500; Epac2 (M-18; Santa Cruz Biotechnology), 1:500.

Techniques: Expressing, Cell Culture, Positive Control, Western Blot, Phospho-proteomics, Control, Two Tailed Test

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 is downregulated in orbital tissues of patients with TAO . ( A – E ) The collected orbital tissue samples from patients with TAO and healthy donors were assessed for histopathological features using H&E and Masson stainings ( A , B ); Epac1 level and distribution using IHC staining ( C ); Epac1 mRNA expression using qRT-PCR ( D ); and Epac1 and vimentin protein levels using immunoblotting ( E ) ( n = 6 or 5). The Student's t -test was conducted to assess comparisons between the two groups, ** P < 0.01 versus the healthy group. TAO, thyroid-associated orbitopathy; H&E, hematoxylin and eosin staining; IHC, immunohistochemical staining; OAT, orbital adipose tissues.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemical staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 is downregulated in OAT and OMT in a TAO mouse model. Mouse OAT and OMT samples were evaluated for histopathological features using H&E and Masson stainings ( A , B ); Epac1 level and distribution using IHC staining ( C ); Epac1 mRNA expression using qRT-PCR ( D ); and Epac1 and vimentin protein levels using immunoblotting ( E ) ( n = 5). The Student's t -test was conducted to assess comparisons between the two groups, ** P < 0.01 versus the Ad-NC group. H&E, hematoxylin and eosin staining; IHC, immunohistochemical staining; OAT, orbital adipose tissues; OMT, orbital muscle tissues; Ad, adenovirus; NC, negative control.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Staining, Immunohistochemical staining, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression attenuates the effects of TGFβ1 on normal and TAO OFs. ( A – G ) Healthy or TAO OFs were transfected with Epac1 -overexpressing plasmid ( Epac1 ), treated with TGFβ1 (10 ng/mL), and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); relative α-SMA and fibronectin expressions using IF staining ( D ); collagen I content in the cell culture supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy OFs; # P < 0.05, ## P < 0.01 versus TAO OFs; && P < 0.01 versus healthy OFs + TGFβ1 + NC; $ P < 0.05, $$ P < 0.01 versus TAO OFs + TGFβ1 + NC. TAO, thyroid-associated orbitopathy; sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay, Migration, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 knockdown amplifies the effects of TGFβ1 on normal and TAO OFs. ( A , B ) Healthy or TAO OFs were transfected with plasmids containing short-hairpin RNA targeting Epac1 (sh- Epac1 #1/#2), treated with TGFβ1 (10 ng/mL), and examined for Epac1 protein levels using immunoblotting; and ( B ) cell viability using CCK-8; sh- Epac1 #1 was selected for the following experiments. ( C – H ) Healthy or TAO OFs were transfected with sh- Epac1 #1, treated with TGFβ1 (10 ng/mL), and examined for cell migration using scratch wound healing and Transwell assays ( C , D ); relative expression of α-SMA and fibronectin using IF staining ( E ); the content of collagen I in cell culture supernatant using ELISA (F); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( G ); and the protein level of Epac1, α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( H ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. * P < 0.05, ** P < 0.01 versus healthy OFs + TGFβ1 + sh-NC; # P < 0.05, ## P < 0.01 versus TAO OFs + TGFβ1 + sh-NC. TAO, thyroid-associated orbitopathy; sh, short hairpin RNA; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Knockdown, Transfection, shRNA, Western Blot, CCK-8 Assay, Migration, Expressing, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression improves TAO-like features in the mouse model. ( A ) A schematic diagram of the TAO model establishment and Epac1 overexpression administration; ( B ) at the end of the modeling, the appearance of mouse eyes was observed; ( C , D ) the histopathological alterations in mouse OAT and OMT samples were evaluated using H&E and Masson stainings. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; NC, negative control; L, left; R, right; H&E, hematoxylin and eosin staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, Virus, Negative Control, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 overexpression decreases TAO-associated markers in vivo. ( A , B ) The protein level of Epac1, α-SMA, CD40, and collagen I in mouse OAT was examined using IHC staining; ( C , D ) the protein level of Epac1, α-SMA, CD40, and collagen I in mouse OMT was examined using IHC staining ( n = 5). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. * P < 0.05, ** P < 0.01 versus healthy; ## P < 0.01 versus TAO + AAV-NC. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; NC, negative control; IHC staining, immunohistochemical staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Over Expression, In Vivo, Immunohistochemistry, Virus, Negative Control, Immunohistochemical staining, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: Epac1 affects the STAT3 signaling. ( A ) A schematic diagram of Epac1’s role in the JAK/STAT3 signaling pathway during the OF activation process. ( B ) TAO OFs transfected with sh- Epac1 or Epacl overexpression vector, treated with TGFβ1, and determined for the protein level of p-STAT3 and STAT3 using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus NC-transfected OFs; ## P < 0.01 versus sh-NC-transfected OFs. ( C ) The protein level of p-STAT3 and STAT3 in TAO mouse OAT and OMT was determined using immunoblotting ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus healthy mice; # P < 0.05, ## P < 0.01 versus TAO + AAV-NC mice. TAO, thyroid-associated orbitopathy; OAT, orbital adipose tissues; OMT, orbital muscle tissues; AAV, adeno-associated virus; sh, short-hairpin RNA; NC, negative control.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Activation Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Virus, shRNA, Negative Control

Journal: Investigative Ophthalmology & Visual Science

Article Title: Epac1 Inhibits Orbital Fibroblast Activation to Ameliorate Thyroid-Associated Orbitopathy-Like Features Through the JAK/STAT Signaling Pathway

doi: 10.1167/iovs.66.9.68

Figure Lengend Snippet: STAT3 mediates the effects of Epac1 on TGFβ1-treated TAO OFs. ( A – G ) Under TGFβ1 treatment, TAO OFs were transfected with sh- Epac1 with or without the JAK-STAT pathway inhibitor Stattic (2 g/mL for 24 hours) and examined for cell viability using CCK-8 ( A ); cell migration using scratch wound healing and Transwell assays ( B , C ); α-SMA and fibronectin expressions using IF staining ( D ); the content of collagen I in cell supernatant using ELISA ( E ); the mRNA expression of Epac1 , α-SMA, vimentin, fibronectin, collagen I, and collagen III using qRT-PCR ( F ); the protein level of α-SMA, vimentin, fibronectin, collagen I, and collagen III using immunoblotting ( G ) ( n = 3). One-way ANOVA with post hoc Tukey's test was used for analyzing comparisons among multiple groups. ** P < 0.01 versus TGFβ1 + sh-NC; ## P < 0.01 versus TGFβ1 + sh- Epac1 + Stattic. sh, short-hairpin; NC, negative control; IF staining, immunofluorescent staining.

Article Snippet: Afterward, the sections were incubated (30 minutes) with 5% normal goat serum in 1% BSA-containing PBS to block the nonspecific immunoglobulin binding sites, followed by incubation (overnight at 4°C) with primary antibodies against Epac1 (DF6922; Affinity Bioscience, Changzhou, China), CD40 (AF5336; Affinity Bioscience), collagen I (14695-1-AP; Proteintech, Wuhan, China), and alpha-smooth muscle actin (α-SMA; 55135-1-AP; Proteintech).

Techniques: Transfection, CCK-8 Assay, Migration, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Negative Control