Whole retinal lysates were collected in lysis buffer containing protease and phosphatase inhibitors. Equal amounts of protein were separated on a precast Tris-glycine gel (Invitrogen) and blotted on nitrocellulose membrane. After blocking in Tris-buffered saline and Tween 20 (TBST; 10 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and 5% (w/v) bovine serum albumin, the membranes were treated with Epac1 (ab109415), Epac 2 (ab193665, Abcam), total nuclear factor kappa beta (NFκB; #4764), phosphorylated NFκB (Ser 536, #3303), phosphorylated IκB (Ser32, #2859), total IκB (#4812, Cell Signaling, Danvers, MA), and beta actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies followed by incubation with secondary antibodies labeled with horseradish peroxidase. Antigen-antibody complexes were detected with a chemiluminescence reagent kit (Thermo Scientific, Pittsburgh, PA), and data were acquired using an Azure C500 (Azure Biosystems, Dublin, CA). Western blot data were assessed using Image Studio Lite software.
Ab193665
Manufactured by Abcam
Ab193665 is a recombinant monoclonal antibody that recognizes the protein of interest. It is designed for use in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Phosphorylated iκb, by Cell Signaling Technology (1 mentions)
Chemiluminescence reagent kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Ab109415, by Abcam (1 mentions)
Total iκb, by Cell Signaling Technology (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Ab44928, by Abcam (1 mentions)
Ab52939, by Abcam (1 mentions)
Ab76238, by Abcam (1 mentions)
Ab75991, by Abcam (1 mentions)
Ab14196, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab4821, by Abcam (1 mentions)
2 protocols using ab193665
1
Epac and NF-κB Regulation in Retina
2
Western Blotting Analysis of Soft Palate
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Western blotting analysis was carried out as reported previously by our lab (20 (link)). Briefly, after the total protein of soft palate tissues was extracted and separated, the samples were transferred onto a polyvinylidene fluoride (PVDF) membrane. Following washing, the membrane was incubated overnight at 4°C with primary antibodies against cAMP (1:1000, Abcam, ab76238), protein kinase A (PKA) (1:1000, Abcam, ab75991), cAMP-regulated guanine nucleotide exchange factor II (Epac2) (1:1000, Abcam, ab193665), rat sarcoma protein (Ras) (1:1000, Abcam, ab52939), c-Jun N-terminal kinase 1/2 (JNK1/2) (1:1000, Abcam, ab4821), Annexiv V (1:500, Abcam, ab14196), GAPDH (1:1000, Abcam, ab8245), and Tubulin (1:1000, Abcam, ab44928). After washing, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at room temperature, followed by color development using a mixed solution. The image analyzer quantitative system was used to quantitatively determine the intensity of the target and reference proteins.
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