epac1  (Addgene inc)

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: Biomedicines

Article Title: GDF6 Alleviates Pathological Cardiac Hypertrophy via AMPKα Signaling Pathway

doi: 10.3390/biomedicines13122935

Figure Lengend Snippet: GDF6 activates AMPKα through cyclic AMP/exchange protein directly activated by cAMP 1 (cAMP/Epac1) pathway. ( A ) The levels of cAMP and protein kinase A (PKA) activity in PE-stimulated NRVMs with or without GDF6 knockdown. ( B ) The levels of cAMP and PKA activity in PE-stimulated NRVMs with or without rhGDF6 treatment. ( C ) Quantification of cell area in rhGDF6-treated NRVMs with or without adenylyl cyclase (AC) inhibition. ( D ) Rap1-GTP and total Rap1 protein levels in PE-stimulated NRVMs treated with si Gdf6 or rhGDF6. ( E ) AMPKα protein levels in PE-stimulated NRVMs with or without Epac1 silence in the presence or absence of rhGDF6. ( F ) Quantification of cell area. ( G ) Epac1 mRNA levels in NRVMs with or without Epac1 silence. ( H ) Quantification of cell area. ( I ) Epac1 mRNA levels in murine hearts with or without Epac1 silence. ( J ) HW/TL in TAC mice with or without Epac1 silence in the presence or absence of AAV9- Gdf6 . ( K ) Quantification of CSA. ( L ) IVSd and IVSs. ( M ) FS, LVEDd, and LVEDs. n = 6 per group. * p < 0.05 versus matched AAV9- Ctrl -injected TAC mice receiving si Ctrl injection, # p < 0.05 versus matched AAV9- Gdf6 -injected TAC mice receiving si Ctrl injection. In ( A – D , G – I ), * p < 0.05 versus the matched groups. In ( E , F ), * p < 0.05 versus matched si Ctrl -transfected NRVMs with DMSO treatment under PE incubation, # p < 0.05 versus matched si Ctrl -transfected NRVMs with rhGDF6 treatment under PE incubation. One-way ANOVA followed by Tukey post hoc test was applied. In ( A , B , D , G – I ), Student’s two-tailed t -test was performed.

Article Snippet: Small interfering RNA against rat Epac1 (si Epac1 , #SR506839), rat GDF6 (si Gdf6 , #SR513148), mouse Epac1 (#SR420934), and rat AMPKα2 (si Ampka2 , #SR508585) were purchased from OriGene (Rockville, MD, USA).

Techniques: Activity Assay, Knockdown, Inhibition, Injection, Transfection, Incubation, Two Tailed Test

Journal: Biomedicines

Article Title: GDF6 Alleviates Pathological Cardiac Hypertrophy via AMPKα Signaling Pathway

doi: 10.3390/biomedicines13122935

Figure Lengend Snippet: GDF6 activates AMPKα through cyclic AMP/exchange protein directly activated by cAMP 1 (cAMP/Epac1) pathway. ( A ) The levels of cAMP and protein kinase A (PKA) activity in PE-stimulated NRVMs with or without GDF6 knockdown. ( B ) The levels of cAMP and PKA activity in PE-stimulated NRVMs with or without rhGDF6 treatment. ( C ) Quantification of cell area in rhGDF6-treated NRVMs with or without adenylyl cyclase (AC) inhibition. ( D ) Rap1-GTP and total Rap1 protein levels in PE-stimulated NRVMs treated with si Gdf6 or rhGDF6. ( E ) AMPKα protein levels in PE-stimulated NRVMs with or without Epac1 silence in the presence or absence of rhGDF6. ( F ) Quantification of cell area. ( G ) Epac1 mRNA levels in NRVMs with or without Epac1 silence. ( H ) Quantification of cell area. ( I ) Epac1 mRNA levels in murine hearts with or without Epac1 silence. ( J ) HW/TL in TAC mice with or without Epac1 silence in the presence or absence of AAV9- Gdf6 . ( K ) Quantification of CSA. ( L ) IVSd and IVSs. ( M ) FS, LVEDd, and LVEDs. n = 6 per group. * p < 0.05 versus matched AAV9- Ctrl -injected TAC mice receiving si Ctrl injection, # p < 0.05 versus matched AAV9- Gdf6 -injected TAC mice receiving si Ctrl injection. In ( A – D , G – I ), * p < 0.05 versus the matched groups. In ( E , F ), * p < 0.05 versus matched si Ctrl -transfected NRVMs with DMSO treatment under PE incubation, # p < 0.05 versus matched si Ctrl -transfected NRVMs with rhGDF6 treatment under PE incubation. One-way ANOVA followed by Tukey post hoc test was applied. In ( A , B , D , G – I ), Student’s two-tailed t -test was performed.

Article Snippet: Small interfering RNA against rat Epac1 (si Epac1 , #SR506839), rat GDF6 (si Gdf6 , #SR513148), mouse Epac1 (#SR420934), and rat AMPKα2 (si Ampka2 , #SR508585) were purchased from OriGene (Rockville, MD, USA).

Techniques: Activity Assay, Knockdown, Inhibition, Injection, Transfection, Incubation, Two Tailed Test