proteintech 10494 1 ap epac1  (Proteintech)

Journal: Scientific Reports

Article Title: Selective antagonism of adenosine A2A receptor reduces hypobaric hypoxia-induced neuroinflammation by inhibiting cGAS-STING pathway

doi: 10.1038/s41598-025-30717-8

Figure Lengend Snippet: KW6002 prevented mtDNA release by inhibiting EPAC1/VDAC1 pathway-mediated opening of MPTP. ( a ) Representative TEM images showing mitochondrial ultrastructure and quantitative analysis of lesions mitochondria in the hippocampal tissues, n = 6/group. ( b ) Cytosolic mtDNA levels of D-loop3, COX1 and mt- ND1 in the hippocampal tissues, n = 4/group. ( c ) Representative images of immunofluorescence staining and quantitative analysis of dsDNA (red)/DAPI (blue), n = 4/group. ( d ) The concentration of cAMP in brain, n = 12/group. ( e , f ) Representative western blot images showing EPAC1 and VDAC1 protein levels in the hippocampal tissues, n = 6/group. ( g ) The levels of inflammatory factors secreted by HY-treated BV2 microglial cells, n = 12/group. ( h ) Representative immunofluorescence staining images showing mitochondrial EPAC1 and VDAC1 levels in HY-induced BV2 cell model, quantitative analysis of levels of EPAC1 and VDAC1, quantitative analysis of colocalization between EPAC1 and VDAC1, n = 8 or 12/group. *** p < 0.001, ** p < 0.01 vs. control group. ### p < 0.001, ## p < 0.01, # p < 0.05 vs. HY group.

Article Snippet: After blocked with 5% non-fat milk, the membranes were incubated successively with specific primary overnight at 4 °C.and secondary antibodies at room temperature for 1 h. (ADORA1 antibody[ R23384 , 1:1000 dilution, Chengdu Zen-Bioscience], ADORA2A antibody[ab79714, 1:1000 dilution, Abcam], ADORA2B antibody[21071-1-AP, 1:1000 dilution, Proteintech], ADORA3 antibody[820199, 1:1000 dilution, Chengdu Zen-Bioscience], ZO-1 antibody[511417, 1:1000 dilution, Chengdu Zen-Bioscience], Occludin antibody [27260-1-AP, 1:1000 dilution, Proteintech], Claudin 1 antibody[13050-1-AP, 1:1000 dilution, Proteintech], PSD95 antibody[20665-1-AP, 1:1000 dilution, Proteintech], SYN1 antibody[20258-1-AP, 1:1000 dilution, Proteintech], ARC antibody[16290-1-AP, 1:1000 dilution, Proteintech], BDNF antibody[66292-1-Ig, 1:1000 dilution, Proteintech], HO-1 antibody[ R22808 , 1:1000 dilution, Chengdu Zen-Bioscience], Nrf2 antibody[12721 S, 1:1000 dilution, Cell Signaling Technology], cGAS antibody[31659, 1:1000 dilution, Cell Signaling Technology], STING antibody[19851-1-AP, 1:2000 dilution, Proteintech], p-STING antibody[PA5-105674, 1:500 dilution, Thermo Fisher Scientific], p-IRF3 antibody[530857, 1:1000 dilution, Chengdu Zen-Bioscience], EPAC1 antibody[12572-1-AP, 1:1000 dilution, Proteintech], VDAC1 antibody[ab154856, 1:1000 dilution, Abcam], GAPDH antibody[ R24404 , 1:5000 dilution, Chengdu Zen-Bioscience], ACTB antibody[R380624, 1:5000 dilution, Chengdu Zen-Bioscience], Tubulin antibody[80713-1-RR, 1:1000 dilution, Proteintech], TOM20 antibody[11802-1-AP, 1:5000 dilution, Proteintech]. secondary antibody Goat Anti-Rabbit IgG H&L(HRP)[511203, 1:5000 dilution, Chengdu Zen-Bioscience], secondary antibody Goat Anti-Mouse IgG H&L(HRP)[511103, 1:5000 dilution, Chengdu Zen-Bioscience].

Techniques: Immunofluorescence, Staining, Concentration Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: Selective antagonism of adenosine A2A receptor reduces hypobaric hypoxia-induced neuroinflammation by inhibiting cGAS-STING pathway

doi: 10.1038/s41598-025-30717-8

Figure Lengend Snippet: Schematic illustration depicting that selective antagonism on ADORA2A reduces HY-induced neuroinflammation by inhibiting cGAS-STING pathway. HY exposure enhanced the level of ADORA2A, which in turn promoted the production of cAMP and cascading mitochondrial levels of EPAC1 and VDAC1. The interaction between EPAC1 and VDAC1 regulated MPTP opening and promoted mtDNA release to cytoplasm, which subsequently initiated cGAS-STING inflammatory pathway. Selective antagonism on ADORA2A restores HY-induced neuroinflammation through negatively regulating above biological process. The illustrative picture was created in BioRender. (yehui, G. (2025) https://BioRender.com/e23u690 ).

Article Snippet: After blocked with 5% non-fat milk, the membranes were incubated successively with specific primary overnight at 4 °C.and secondary antibodies at room temperature for 1 h. (ADORA1 antibody[ R23384 , 1:1000 dilution, Chengdu Zen-Bioscience], ADORA2A antibody[ab79714, 1:1000 dilution, Abcam], ADORA2B antibody[21071-1-AP, 1:1000 dilution, Proteintech], ADORA3 antibody[820199, 1:1000 dilution, Chengdu Zen-Bioscience], ZO-1 antibody[511417, 1:1000 dilution, Chengdu Zen-Bioscience], Occludin antibody [27260-1-AP, 1:1000 dilution, Proteintech], Claudin 1 antibody[13050-1-AP, 1:1000 dilution, Proteintech], PSD95 antibody[20665-1-AP, 1:1000 dilution, Proteintech], SYN1 antibody[20258-1-AP, 1:1000 dilution, Proteintech], ARC antibody[16290-1-AP, 1:1000 dilution, Proteintech], BDNF antibody[66292-1-Ig, 1:1000 dilution, Proteintech], HO-1 antibody[ R22808 , 1:1000 dilution, Chengdu Zen-Bioscience], Nrf2 antibody[12721 S, 1:1000 dilution, Cell Signaling Technology], cGAS antibody[31659, 1:1000 dilution, Cell Signaling Technology], STING antibody[19851-1-AP, 1:2000 dilution, Proteintech], p-STING antibody[PA5-105674, 1:500 dilution, Thermo Fisher Scientific], p-IRF3 antibody[530857, 1:1000 dilution, Chengdu Zen-Bioscience], EPAC1 antibody[12572-1-AP, 1:1000 dilution, Proteintech], VDAC1 antibody[ab154856, 1:1000 dilution, Abcam], GAPDH antibody[ R24404 , 1:5000 dilution, Chengdu Zen-Bioscience], ACTB antibody[R380624, 1:5000 dilution, Chengdu Zen-Bioscience], Tubulin antibody[80713-1-RR, 1:1000 dilution, Proteintech], TOM20 antibody[11802-1-AP, 1:5000 dilution, Proteintech]. secondary antibody Goat Anti-Rabbit IgG H&L(HRP)[511203, 1:5000 dilution, Chengdu Zen-Bioscience], secondary antibody Goat Anti-Mouse IgG H&L(HRP)[511103, 1:5000 dilution, Chengdu Zen-Bioscience].

Techniques:

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

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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: The used primary antibodies were α-SMA (55135-1-AP; Proteintech), Collagen I (14695-1-AP; Proteintech), Vimentin (10366-1-AP; Proteintech), Epac1 (DF6922; Affinity), fibronectin (15613-1-AP; Proteintech), collagen III (22734-1-AP; Proteintech), phosphorylated STAT3 (CSB-RA022812A727phHU; Cusabio, Wuhan, China), STAT3 (10253-2-AP; Proteintech), FABP4 (12802-1-AP; Protientech), and β-actin (CSB- MA000187 ; Cusabio).

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

Journal: Journal of Translational Medicine

Article Title: EphrinB2 alleviates tubulointerstitial fibrosis in diabetic kidney disease

doi: 10.1186/s12967-025-06852-1

Figure Lengend Snippet: Effects of EphrinB2 overexpression on Epac1-Rap1 Pathway in diabetic nephropathy mouse. A Immunofluorescence staining of EphrinB2 (red) and Epac1 (green) in kidney tissues from different experimental groups. DAPI (blue) was used for nuclear staining. Merged images show colocalization. Scale bar = 50 μm. B , C Quantification of EphrinB2 fluorescence intensity ( B ) and Epac1 fluorescence intensity ( C ) in different groups. ( D ) Immunohistochemical staining of Epac1, Rap1, and PGC-1α in kidney tissues from control (CTL) and STZ-induced diabetic mice, with or without EphrinB2 overexpression. Scale bar = 50 μm. E–G Quantification of Epac1-positive area ( E ), Rap1-positive area ( F ), and PGC-1α-positive area ( G ) in different groups. ( H ) Western blot analysis of Epac1 and Rap1 protein expression in kidney tissues from wild-type (WT), STZ 12-week (STZ12W), and STZ 16-week (STZ16W) mice. β-actin was used as a loading control. I Densitometric quantification of Epac1 and Rap1 protein levels, normalized to β-actin, in WT, STZ12W, and STZ16W groups. All results were presented as the mean ± SD, n = 3/group. * p < 0.05, ** p < 0.01

Article Snippet: Epac1 , 1:1000 , Santa Cruz , SC-28366.

Techniques: Over Expression, Immunofluorescence, Staining, Fluorescence, Immunohistochemical staining, Control, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: EphrinB2 alleviates tubulointerstitial fibrosis in diabetic kidney disease

doi: 10.1186/s12967-025-06852-1

Figure Lengend Snippet: EphrinB2 Overexpression Restores Mitochondrial Dynamics and Reduces Fibrosis via the Epac1-Rap1 Pathway. A Western blot analysis of proteins involved in mitochondrial dynamics and function, including p-Drp1, Drp1, Opa1, Mfn1, Mfn2, PGC-1α, Rap1, and Epac1 in control and STZ-treated mice with or without EphrinB2 overexpression (EphrinB2-OE). β-actin serves as a loading control. B Quantification of protein expression for EPAC, Rap1, and PGC-1α showing relative changes across different experimental groups. C Quantification of mitochondrial fusion proteins Mfn1, Mfn2, and Opa1 expression levels. D Ratio of phosphorylated Drp1 (p-Drp1) to total Drp1 indicating changes in mitochondrial fission upon different treatments. E Western blot analysis for the same set of proteins in cultured cells under low glucose (LG) and high glucose (HG) conditions, with or without EphrinB2-OE. F Quantification of EphrinB2 and PGC-1α protein levels under different glucose conditions. G Bar graphs showing relative protein levels of EPAC and Rap1 in cultured cells under high glucose conditions. H Expression levels of mitochondrial fusion proteins Mfn1, Mfn2, and Opa1 under different experimental setups. I Analysis of p-Drp1 levels in cells exposed to high glucose with or without EphrinB2-OE. J Western blots for fibrosis markers including Fibronectin and α-SMA, along with mitochondrial dynamics proteins under various conditions. K – L Quantification of EPAC1, Rap1, and PGC-1α levels under the influence of high glucose and different treatment modifiers. ( M ) . Expression levels of mitochondrial fusion proteins under low and high glucose conditions with additional treatment conditions. N Quantitative analysis of α-SMA levels, showing the effect of EphrinB2-OE and its modifiers on fibrotic response in high glucose conditions. O , P Statistical analysis of the ratio of p-Drp1 to Drp1 and the expression levels of fibrosis markers α-SMA and Fibronectin, indicating the regulatory impact of EphrinB2 on mitochondrial dynamics and cellular fibrosis under diabetic conditions. All results were presented as the mean ± SD, n = 3/group. * p < 0.05, ** p < 0.01

Article Snippet: Epac1 , 1:1000 , Santa Cruz , SC-28366.

Techniques: Over Expression, Western Blot, Control, Expressing, Cell Culture

Journal: Journal of Translational Medicine

Article Title: EphrinB2 alleviates tubulointerstitial fibrosis in diabetic kidney disease

doi: 10.1186/s12967-025-06852-1

Figure Lengend Snippet: EphrinB2 Overexpression Stabilizes Epac1 Protein Under High-Glucose Conditions. A Immunofluorescence staining for EphrinB2 (red) and Epac1 (green) with nuclei stained using DAPI (blue) in cells under low glucose (LG) and high glucose (HG) conditions, with or without EphrinB2 overexpression (EphrinB2-OE). Scale bar, 50 µm. B Quantification of fluorescence intensity for EphrinB2 and Epac1 under the described conditions. C Western blot analysis of Epac1 protein stability with cycloheximide (CHX) treatment over time (0, 4, 8, 12 h) in cells transfected with either vector control or EphrinB2-OE. D Co-immunoprecipitation (co-IP) showing the interaction between EphrinB2 and Epac1 in LG and HG conditions, with or without EphrinB2-OE. E Graph showing the relative expression levels of Epac1 protein over time with CHX treatment in vector control and EphrinB2-OE conditions, demonstrating changes in protein stability. All results were presented as the mean ± SD, n = 3/group. * p < 0.05, ** p < 0.01

Article Snippet: Epac1 , 1:1000 , Santa Cruz , SC-28366.

Techniques: Over Expression, Immunofluorescence, Staining, Fluorescence, Western Blot, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing