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Epac1

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Epac1 is a cAMP-activated guanine nucleotide exchange factor (GEF) that acts as a regulator of Rap1 and Rap2 small GTPases. It plays a role in various cellular processes, including cell-cell adhesion, cell-matrix adhesion, and cell migration.

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Lab products found in correlation

β actin, by Santa Cruz Biotechnology (9 mentions) Azure c500, by Azure Biosystems (6 mentions) Tris glycine gel, by Thermo Fisher Scientific (6 mentions) Chemiluminescence reagent kit, by Thermo Fisher Scientific (5 mentions) Il 1β, by Abcam (5 mentions) Chemilluminescence reagent kit, by Thermo Fisher Scientific (4 mentions) Nlrp3, by Abcam (4 mentions) Occludin, by Abcam (3 mentions) Traf6, by Abcam (3 mentions) Hmgb1, by Abcam (3 mentions) Cleaved caspase 1, by Abcam (3 mentions) Myd88, by Abcam (2 mentions) Azure c500 machine, by Azure Biosystems (2 mentions) Pre cast tris glycine gel, by Thermo Fisher Scientific (2 mentions) Tnf α, by Abcam (2 mentions) Image studio light software, by LI COR (1 mentions) Vascular endothelial growth factor (vegf), by Abcam (1 mentions) Serca2, by Cell Signaling Technology (1 mentions) P pka, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions)

13 protocols using epac1

1

Western Blot Analysis of Protein Expression

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Cell culture lysates were collected into lysis buffer with both protease and phosphatase inhibitors. Equal amounts of protein were separated using pre-cast tris–glycine gels (Invitrogen, Carlsbad, CA), and blotted onto nitrocellulose membranes. After blocking in TBST (10 mM Tris–HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes were treated with an Epac1, TLR4, MyD88, IRAK, TRAF6, TRAM1, IRF3, occludin, and ZO-1 (Abcam, Cambridge, MA) and beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies tagged with horseradish peroxidase. Antigen–antibody complexes were visualized with an Azure C500 machine (Azure Biosystems, Dublin, CA) after application of a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Western blot band densities were measured using Image Studio Lite software.
Liu L., Jiang Y, & Steinle J.J. (2021). Forskolin regulates retinal endothelial cell permeability through TLR4 actions in vitro. Molecular and cellular biochemistry, 476(12), 4487-4492.
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2

Western Blot Analysis of Retinal Proteins

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After appropriate treatments and rinsing with cold PBS, REC or whole retinal lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein from the cell extracts were separated on the precast tris-glycine gel (Invitrogen), blotted onto a nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (wt/vol) BSA, the membranes was treated with Epac1, PKCzeta, Occludin, ZO-1, VEGF (all from Abcam, San Francisco, CA, USA), or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA, USA). Data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA, USA). Western blot analyses were done using Image Studio Light software (LI-COR, Lincoln, NE, USA).
Jiang Y., Liu L, & Steinle J.J. (2017). Compound 49b Regulates ZO-1 and Occludin Levels in Human Retinal Endothelial Cells and in Mouse Retinal Vasculature. Investigative Ophthalmology & Visual Science, 58(1), 185-189.
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3

Western Blot Analysis of Cardiac Proteins

Cited 2 times
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Western blot analysis was performed as we described previously [39 (link)–42 (link)]. Protein samples of the cardiomyocytes and fibroblasts isolated from the mouse hearts were prepared in RIPA lysis buffer. 30μg of total proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad). Then the membranes were incubated overnight (4°C) with a primary antibody against AC4 (Sigma, SAB4300752, 1:1000), PKA (Cell Signaling, #5842, 1:1000), P-PKA (Cell Signaling, #5661, 1:1000), PLN (Cell Signaling, #14562, 1:1000), P-PLN (Cell Signaling, #8496, 1:1000), SERCA2 (Cell Signaling, #9580, 1:1000), Cleaved-caspase 3 (Cell Signaling, #9661, 1:1000), Epac1 (Abcam, ab109415, 1:1000), GAPDH (Santa Cruz, sc-32233, 1:1000). Goat anti-mouse or goat anti-rabbit horseradish peroxidase (1:2000–1:5,000; Santa Cruz Biotechnology) was used as a secondary antibody and incubated for 1 hour at room temperature. Specific proteins were detected by chemiluminescent methods using Clarity™ western ECL substrate (Bio-Rad, Hercules, CA). Protein abundance on western blots was quantified by densitometry using Image lab software (Bio-Rad, Hercules, CA).
Chen K., Wang S, & Sun Z. (2022). In Vivo Cardiac-specific Expression of Adenylyl Cyclase 4 Gene Protects against Klotho Deficiency-induced Heart Failure. Translational research : the journal of laboratory and clinical medicine, 244, 101-113.
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4

Quantitative Immunoblotting of Retinal Proteins

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Retinal cell lysates were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated onto a pre-cast tris-glycine gel (Invitrogen, Carlsbad, CA), blotted onto nitrocellulose membrane and blocked with TBST (10mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA. Membranes were treated with HMGB1, IGFBP-3, Epac1(Abcam, Cambridge, MA) or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with horseradish peroxidase labeled secondary antibodies. Antigen-antibody complexes were detected by chemilluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) and data was acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.
Liu L., Patel P, & Steinle J.J. (2018). PKA regulates HMGB1 through activation of IGFBP-3 and SIRT1 in human retinal endothelial cells cultured in high glucose. Inflammation research : official journal of the European Histamine Research Society ... [et al.], 67(11-12), 1013-1019.
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5

Western Blotting of Retinal Proteins

Cited 1 time
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Western blotting was done on REC or whole retinal lysates as we have done previously (Liu et al., 2017a (link); Zhang et al., 2012 (link)). Primary antibodies used were Epac1, NLRP3, P2X7R, IL-1β, cleaved caspase 1 (Abcam, Cambridge, MA) and beta actin (Santa Cruz, Santa Cruz, CA)
Liu L., Jiang Y, & Steinle J.J. (2022). Epac1 and PKA regulate of P2X7 and NLRP3 inflammasome proteins in the retinal vasculature. Experimental eye research, 218, 108987.
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6

Protein Quantification and Characterization

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Cells were harvested using standard techniques (cell lysis buffer containing protease inhibitors). Protein was separated by SDS PAGE using a 4–15% Tris-HCl gel and transferred to nitrocellulose membranes using a Bio-Rad Trans-Blot Turbo rapid transfer system (Bio-Rad, Hercules, CA). Primary antibodies used include BDNF, Epac1, Epac2 (Abcam, Cambridge, MA), and β-actin (Sigma-Aldrich, St. Louis, MO). Membranes were imaged on a Li-Cor OdysseyXL system and densitometry was quantified with Image Studio software. Protein blots were normalized to β-actin.
Thompson M.A., Britt RD J.r., Kuipers I., Stewart A., Thu J., Pandya H.C., McFarlane P., Pabelick C.M., Martin R.J, & Prakash Y.S. (2015). cAMP-Mediated Secretion of Brain-Derived Neurotrophic Factor in Developing Airway Smooth Muscle. Biochimica et biophysica acta, 1853(10 0 0), 2506-2514.
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7

Western Blot Analysis of Cell Signaling

Cited 2 times
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Western blotting was performed as previously described [12 (link),13 (link)]. Briefly, cell lysates or whole retina lysates were separated onto precast tris-glycine gels (Invitrogen, Carlsbad, CA) and then blotted onto nitrocellulose membrane. After blocking in TBST, membranes were treated with Epac1, TNFAIP3, TRAF6, TNFα, phosphorylated and total NF-kB, phosphorylated and total IkB, occludin, ZO-1 (Abcam, Cambridge, MA), or beta actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies. Secondary species-appropriate antibodies labeled with horseradish peroxidase were applied after the primary antibodies. Chemiluminescence (Thermo Scientific, Pittsburgh, PA) was used to visualize antigen–antibody complexes. Images were acquired on an Azure C500 (Azure Biosystems, Dublin, CA), and optical densities were measured using Image Studio Lite software (Lincoln, NE).
Liu L., Jiang Y, & Steinle J.J. (2022). TNFAIP3 is anti-inflammatory in the retinal vasculature. Molecular Vision, 28, 124-129.
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8

Western Blot Analysis of Retinal Proteins

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Whole retinal lysates or REC were collected into lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were loaded and separated onto precast Tris-glycine gels (Invitrogen, Carlsbad, CA) and then blotted onto a nitrocellulose membrane. The membranes were blocked in TBST (10 mM Tris-HCl buffer, pH 8.0; 150 mM NaCl, and 0.1% Tween 20) and 5% BSA and then incubated with corresponding primary antibodies, followed by incubation with secondary antibodies labeled with horseradish peroxidase. Primary antibodies used included Epac1, IL-1β, Rap 1 (Abcam, Cambridge, MA), phosphorylated p38 MAPK, total p38 MAPK, phosphorylated JNK, total JNK, phosphorylated insulin receptor (Tyr 1150/1151), total insulin receptor, phosphorylated Akt (Ser473), total Akt, Akt2, phosphorylated IRS-1 (Ser307), total IRS-1 (Cell Signaling Corp, Danvers, MA), and beta-actin (Santa Cruz, CA). Phosphorylated proteins were compared to total protein levels, and the other proteins were compared to beta-actin. Antigen-antibody complexes were identified using a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA). Imaging was performed using a C500 (Azure Biosystems, Dublin, CA).
Curtiss E., Jiang Y., Liu L., Hawthorne C., Zhang J, & Steinle J.J. (2018). Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines. Mediators of Inflammation, 2018, 3809092.
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9

Immunoblotting Analysis of Retinal Proteins

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Whole retinal lysates from mice or cell culture lysates were collected into lysis buffer with protease and phosphatase inhibitors. Equal amounts of protein separated onto a pre-cast tris-glycine gel (Invitrogen, Carlsbad, CA) were blotted onto the nitrocellulose membrane. After blocking in TBST (10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and 5% (w/v) BSA, membranes are treated with Epac1, phosphorylated PKR, total PKR, NLRP3, cleaved caspase 1, and IL-1β (Abcam, Cambridge, MA) or beta-actin (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. A chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA) was used to visualize antigen–antibody complexes. Images are acquired on an Azure C500 (Azure Biosystems, Dublin, CA) and optical densities were determined using Image Studio Lite software.
Jiang Y, & Steinle J.J. (2019). Epac1 inhibits PKR to reduce NLRP3 inflammasome proteins in retinal endothelial cells. Journal of Inflammation Research, 12, 153-159.
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10

Western Blot Analysis of Epac1, AMPK, SIRT1, HMGB1

Cited 2 times
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Western blotting was carried out as previously described.7 (link),8 (link) Briefly, cell lysates or whole retina lysates were separated onto precast Invitrogen Tris-Glycine Gels (Thermo Fisher Scientific) and then blotted onto nitrocellulose membrane. After blocking in Tris-Buffered Saline Tween-20, membranes were treated with Epac1, phosphorylated AMPK (Tyr172), total AMPK, SIRT1, HMGB1 (Abcam, Cambridge, MA, USA), or beta-actin (Santa Cruz Biotechnology, Dallas, CA, USA) primary antibodies. Secondary antibodies labeled with horseradish peroxidase were also used. A chemiluminescence reagent kit (Thermo Fisher Scientific) was used to visualize antigen–antibody complexes. Images were acquired on an Azure C500 (Azure Biosystems, Dublin, CA, USA), and optical densities were determined using Image Studio Lite software (LI-COR Biosciences, Lincoln, NE, USA).
Jiang Y, & Steinle J.J. (2020). Epac1 Requires AMPK Phosphorylation to Regulate HMGB1 in the Retinal Vasculature. Investigative Ophthalmology & Visual Science, 61(11), 33.
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