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.
Ab109415
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab109415 is a recombinant protein used as a control in various experimental procedures. The product is made using a proprietary method and is supplied in a lyophilized format.
Automatically generated - may contain errors
Lab products found in correlation
Ls c180179, by LSBio (2 mentions)
Chemiluminescence reagent kit, by Thermo Fisher Scientific (1 mentions)
Ab193665, by Abcam (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Phosphorylated iκb, by Cell Signaling Technology (1 mentions)
Total iκb, by Cell Signaling Technology (1 mentions)
Azure c500, by Azure Biosystems (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Ab32096, by Abcam (1 mentions)
Ab14404, by Abcam (1 mentions)
Ab170939, by Abcam (1 mentions)
Ab32373, by Abcam (1 mentions)
Ab14607, by Abcam (1 mentions)
Ab171750, by Abcam (1 mentions)
Ab75991, by Abcam (1 mentions)
Ab40855, by Abcam (1 mentions)
Ab31387, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab53100, by Abcam (1 mentions)
5 protocols using ab109415
1
Epac and NF-κB Regulation in Retina
2
Protein Expression Analysis of Fibrosis Markers
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The protein levels of α-SMA, COL1A1, Smad2, p-Smad2, PED7A, PED4A, PKA, p-PKA, total Ras-proximate-1 (Rap1), Guanosine-5′-triphosphate (GTP)-Rap1, and exchange protein directly activated by cAMP 1 (Epac1), cAMP-response element-binding protein (CREB) and p-CREB were examined by immunoblotting following the methods described before (Liu et al., 2018 (link)) using the antibodies listed below: anti-α-SMA (ab5694, Abcam), anti-COL1A1 (ab34710, Abcam), anti-Smad2 (ab40855, Abcam), anti-p-Smad2 (ab53100, Abcam), anti-PDE4A (ab14607, Abcam), anti-PDE4B (ab170939, Abcam), anti-PDE4C (ab170939, Abcam), anti-PDE4D (ab171750, Abcam), anti-PKA (BS-0520R, Woburn, MA, United States), p-PKA (ab75991, Abcam), anti-GTP-Rap1 (ab32373, Abcam), anti-Rap1 (ab14404, Abcam), anti-Epac1 (ab109415, Abcam), anti-CREB (ab31387, Abcam), anti-p-CREB (ab32096, Abcam), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam) and then with HRP-conjugated secondary antibody. Enhanced chemilumescent (ECL) substrates (Millipore, MA, United States) were used for signals visualization using GAPDH as an endogenous protein for normalization.
3
Immunostaining of Epac1, Piezo2, and Serotonin in Colon Tissues
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The distal colon tissues and DRGs were fixed with 4% paraformaldehyde for 24 h, and cross-sections of the colonic tissues were obtained after being embedded in paraffin. The sections were stained with diaminobenzidine for 15 s. All sections were blocked with 5% goat serum and incubated with the primary antibody at 4°C overnight. The sections were then washed three times in phosphate-buffered saline (PBS) and incubated at room temperature with the secondary antibody for 1 h. For immunofluorescence analysis, the nuclei were counterstained with 4’,6‐diamino‐2‐phenylindole (DAPI). For primary antibodies, we used rabbit anti-Epac1 antibody (1:50, ab109415, Abcam, Cambridge, UK) or polyclonal rabbit anti−human Piezo2/FAM38B antibody (1:200, LS-C180179, LSBio, Seattle, USA) or serotonin antibody (1:200, sc-58031, Santacruz Biotech, Texas, USA). For secondary antibodies, we used goat anti-rabbit IgG H&L (HRP) (1:1000, ab205718, Abcam, Cambridge, UK), and goat anti-rabbit IgG H&L (Alexa Fluor® 488) (1:1000, ab150077, Abcam, Cambridge, UK). The samples were then viewed under a fluorescence microscope (Olympus, Japan).
4
Western Blot Analysis of Synaptic Proteins
5
Quantitative Protein Expression Analysis
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The colonic tissues and DRGs were homogenized with RIPA lysis buffer. After cooling in an ice-water bath, the tissues were immediately centrifuged at 10000 rpm and 4°C for 10 minutes, and the supernatant was removed for testing. Enhanced BCA Protein Assay Kit (P0010) was used to measure protein concentrations. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature before being probed with rabbit anti-Epac1 antibody (1:1000, ab109415, Abcam, Cambridge, UK) or polyclonal rabbit anti−human Piezo2/FAM38B antibody (1:1000, LS-C180179, LSBio, Seattle, US) or rabbit anti-HTR3A antibody (1:500, 10443-1-AP, Proteintech, Chicago, USA) at 4°C overnight. After washing the membranes, they were subsequently incubated with goat anti-rabbit IgG (H+L) HRP (1:5000, S0001, Affinity Biosciences, Jiangsu, China) at room temperature for 1 h. Protein expression was quantified using Image J (National Institutes of Health, Bethesda, Maryland, USA).
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