The antibodies were used as follows: mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, MAB374, Merck, 1:10000); rabbit polyclonal phosphorylated form of cAMP-dependent protein kinase (pPKA (Thr197), 44988A, Thermo Fisher Scientific, 1:1000); rabbit polyclonal to the recombinant fragment corresponding to a region within amino acids 1 and 351 of PKA catalytic subunit alpha (PA5-21842, Invitrogen, 1:1000); mouse monoclonal to phosphorylated form of cAMP response element-binding protein (CREB (Ser 133), 9196S, Cell Signaling, 1:500); rabbit monoclonal to cAMP response element-binding protein (CREB, 9197S, Cell Signaling, 1:1000); rabbit polyclonal to adenylate cyclase 3 (AC3, PA1-31191, Invitrogen, 1:250); rabbit polyclonal to adenylate cyclase 7 (AC7, PA5-103390, Thermo Fisher Scientific, 1:500); mouse monoclonal to A-kinase anchoring protein 9 (AKAP9, ab32679, Abcam, 1:125); goat monoclonal to A-kinase anchoring protein 9 (AKAP9, ab31307, Abcam, 1:200); rabbit monoclonal to cAMP-specific 3′,5′-cyclic phosphodiesterase 4D (PDE4D, ab171750, Abcam, 1:1000); HRP rabbit polyclonal to β-tubulin (ab21058, Abcam, 1:10000); rabbit polyclonal to MVI (25–6791, Proteus, 1:500); goat anti-mouse IgG antibody, HRP conjugate (AP308P, Millipore, 1:10000); goat anti-rabbit IgG antibody, HRP conjugate (AP307P, Millipore, 1:10000).
Ab171750
Manufactured by Abcam
Sourced in United States
Ab171750 is a mouse monoclonal antibody that targets GAPDH, a key enzyme involved in glycolysis. It can be used for the detection of GAPDH in various applications such as Western blotting, immunocytochemistry, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab32096, by Abcam (2 mentions)
Ab170939, by Abcam (2 mentions)
Ab75991, by Abcam (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab31307, by Abcam (1 mentions)
Ap308p, by Merck Group (1 mentions)
Ap307p, by Merck Group (1 mentions)
Ab21058, by Abcam (1 mentions)
Creb 9197s, by Cell Signaling Technology (1 mentions)
Mab374, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Hyperpage prestained protein marker, by Meridian Bioscience (1 mentions)
A5441, by Merck Group (1 mentions)
Immobilon, by Merck Group (1 mentions)
Ab14404, by Abcam (1 mentions)
Ab32373, by Abcam (1 mentions)
Ab14607, by Abcam (1 mentions)
Ab109415, by Abcam (1 mentions)
Ab40855, by Abcam (1 mentions)
Ab31387, by Abcam (1 mentions)
5 protocols using ab171750
1
Antibody-based Protein Expression Analysis
2
Western Blot Analysis of PDE4B and PDE4D
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The protein lysates were resolved using sodium dodecyl sulphate-polyacrilamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Immobilon, Merck-Millipore; Burlington MA, USA, IPVH00010). The blots were blocked with 5% non-fat dry milk in Tris-buffered saline, 0.05% Tween 20 (TBST) buffer at room temperature for 1 h. The membranes were then incubated overnight with antibodies directed against PDE4B (NBP2-01171, Novus Biologicals, Minneapolis, MN, USA), PDE4D (ab171750, Abcam, Cambridge, UK) and ß-actin (A5441, Sigma-Aldrich). The appropriate horseradish peroxidase-conjugated secondary antibodies (Dako Products, Agilent, Santa Clara, CA, USA) and the LuminataTM Western horseradish peroxidase (HRP) Substrate (Immobilon, Merck-Millipore) were used to detect the bound antibodies. The size of the detected proteins was estimated using protein molecular-mass standards (Hyperpage Prestained Protein Marker; Bioline, Paris, France). ß-actin was used to verify the equal loading of the protein on each lane.
3
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.
4
Rolipram and Nitric Oxide Regulation
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Rolipram was purchased from Medchem Express. Tetrodotoxin (TTX), Nω‐Nitro‐L‐arginine (L‐NNA), Rp‐Adenosine 3′,5′‐cyclic monophosphorothioate triethylammonium salt hydrate (Rp‐cAMPS) were all purchased from Sigma‐Aldrich. Primary antibodies were used as follows: the rabbit anti‐PDE4D(ab171750, Abcam), the rabbit anti‐p‐CREB (ab32096, Abcam), and the rabbit anti‐PKA(ab75991, Abcam), the rabbit anti‐nNOS (4231, Cell signaling), BCA protein assay kit (Beyotime).
The Nitric Oxide Assay kit was bought from Beyotime (S0021S, Beyotime), and cAMP ELISA kit was from Elabscience(E‐EL‐0056c, Elabscience).
TTX, L‐NNA, and Rp‐cAMPS were dissolved in Tyrode's buffer. In contraction recordings of colonic muscle strips in vitro, the dimethylsulfoxide (DMSO) concentration was <0.01%, and the rest of the diluent for Rolipram was Tyrode's buffer. In experiments of intraperitoneal administration in rats, the diluent for Rolipram was DMSO (the added concentration of DMSO was 10%), 70% PEG300, 10% Tween‐80, and 10% saline, so the final DMSO concentration was 1%.
The Nitric Oxide Assay kit was bought from Beyotime (S0021S, Beyotime), and cAMP ELISA kit was from Elabscience(E‐EL‐0056c, Elabscience).
TTX, L‐NNA, and Rp‐cAMPS were dissolved in Tyrode's buffer. In contraction recordings of colonic muscle strips in vitro, the dimethylsulfoxide (DMSO) concentration was <0.01%, and the rest of the diluent for Rolipram was Tyrode's buffer. In experiments of intraperitoneal administration in rats, the diluent for Rolipram was DMSO (the added concentration of DMSO was 10%), 70% PEG300, 10% Tween‐80, and 10% saline, so the final DMSO concentration was 1%.
5
Protein Extraction and Western Blot Analysis
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Cells were homogenized in lysis buffer containing: 1 mmol/L EGTA, 10 mmol/L HEPES, 150 mmol/L NaCl, 300 mmol/L sucrose, 1% Triton-X, and phosphatase and protease inhibitors (Roche, Welwyn Garden City, UK). Protein concentration was determined using Pierce BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Protein lysates were mixed with 4 × SDS sample buffer containing: 200 mmol/L Tris-HCl, 8% SDS, 40% Glycerol, 0.4% Bromophenol blue, and 40 mmol/L DTT, and boiled at 95 °C for 5 min. Proteins (20 µg of total protein per lane) were size separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. For immunodetection primary GAPDH (5G4, dilution 1:160,000, Bio Trend, Koln, Germany), PDE1C (generous gift from C. Yan, dilution 1:1000), PDE2A (PD2A-101AP, dilution 1:500 Fabgennix, Frisco, TX, USA), PDE3A (generous gift from C. Yan, dilution 1:1000), PDE4B (ab170939, dilution 1:2500, Abcam, Cambridge, UK), PDE4D (ab171750, dilution 1:2500, Abcam, Cambridge, UK), and PDE5A (generous gift from S. Rybalkin, dilution 1:100) were used.
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