Western blotting was carried out as previously described (Newson et al., 2014 (link)). Briefly, cells from peritoneal washouts or ex vivo culture were lysed in RIPA buffer with protease inhibitors (both Sigma) and the protein concentration determined by Bradford assay (Bio-Rad). Ten micrograms of protein were separated by SDS-PAGE (National Diagnostics). Separated proteins were transferred onto a polyvinylidene fluoride membrane (Immobilon; Millipore) and incubated with COX-1, COX-2, mPGES-1, mPGE-2, EP1-4 (Cayman Chemical), β-actin, and GAPDH (Sigma) overnight in block buffer (Tris-HCL, 1% Tween-20, 1% BSA [Sigma], and 5% nonfat milk [Marvel]). Blots were washed and incubated with HRP-conjugated antibodies (Santa Cruz Biotechnology) for 1 hr at room temperature in blocking buffer. Specific proteins were visualized by enhanced chemiluminescence (ECL) hyperfilm.
Cox 1
Manufactured by Cayman Chemical
Sourced in United States
COX-1 is a lab equipment product that serves as a cyclo-oxygenase-1 enzyme. It is used for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Cox 2, by Cayman Chemical (7 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Hrp conjugated antibodies, by Santa Cruz Biotechnology (1 mentions)
Immobilon, by Merck Group (1 mentions)
Mpges 1, by Cayman Chemical (1 mentions)
Sds page, by National Diagnostics (1 mentions)
Ep1 4, by Cayman Chemical (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Enhanced chemiluminescent detection, by PerkinElmer (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Ptgs1, by Cayman Chemical (1 mentions)
Cyclin a, by Cell Signaling Technology (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
12 protocols using cox 1
1
Western Blotting for Inflammation Mediators
2
Western Blot Analysis of Cellular Proteins
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After experimental periods, cultured cells were washed twice with PBS and solubilized in lysis buffer containing 40 mM Tris buffer (pH 7.5), 8 M urea, 4% CHAPS, 1 mM PMSF, 1 mM Na3VO4, 1 mM dithiothreitol, and a protease inhibitor kit (BM, Germany). Protein was quantified by a Biorad protein assay reagent. Aliquots of proteins (5 μg) of the cell lysates were analyzed by western blot, using SDS-PAGE (8% or 12% gel), as previously described [35 (link), 41 (link)]. After electrophoresis, gels were transferred to PVDF membranes (Millipore Corp., USA) and incubated overnight at 4 °C with antibodies against PGIS (rabbit, dilution 1/3000), COX-1, COX-2 (rabbit, dilution 1/5000, Cayman) or β-actin (1:5000; Santa Cruz Biotech, USA) followed by a HRP-conjugated secondary antibody (dilution 1/2000, Jackson Lab) for 1 h at room temperature. Immunoreactivity was visualized by enhanced chemiluminescent detection (Perkin Elmer Co, USA).
3
Cyclooxygenase Inhibition Assay
4
Antibody Panel for Cellular Signaling
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Antibody against PPARα was from Abcam. P-p65, P65, AKT, P-AKT, P-p44/42, Erk2, FASN, ACC1, P53, P21, cyclin A, cyclin E, and GAPDH antibodies were from Cell Signaling Technology, Inc., Danvers, MA. Antibodies used against β-actin, FASN tyrosine phosphorylation (PT66), and tubulin, were from Sigma. 5-LO, LTA4H, COX-1 and COX-2 antibodies were from Cayman Chemicals, Ann Arbor, MI.
5
Molecular Mechanisms of PPAR and SIRT1
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All the reagents, including insulin-transferrin-sodium selenite media supplement (ITS), recombinant human IL-1β, GW0742, nicotinamide, and EX527, were purchased from Sigma Chemical Company. Adenoviruses expressing PPARβ/δ (Ad-PPARβ/δ) and TTA (Ad-TTA) were kindly provided by Professor Nanping Wang of the East China Normal University Health Science Center, Shanghai, China. Adenoviruses expressing SIRT1 (Ad-SIRT1), SIRT1 siRNA (Ad-siSIRT1), and the control adenoviruse (Ad-siControl) were generously provided as gifts by Professor Yongsheng Chang of the Beijing Union Medical College, Beijing, China. Antibodies, including β-actin (Santa Cruz sc-47778, Dallas, TX, USA), COX-1 (Cayman Chemical 160109, Ann Arbor, MI, USA), COX-2 (Cayman Chemical 160106), PPARα (Santa Cruz sc-1982), PPARγ (Santa Cruz sc-1981), PPARβ/δ (Abcam ab8937, Cambridge, UK), and SIRT1 (Abcam ab32441) were used in the present study.
6
Licochalcones Inhibit Prostaglandin Synthesis
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Licochalcones were provided by Research Laboratory of Minophagen Pharmaceutical Co. Ltd. (Zama, Japan). Prostaglandin (PG) E2, d4-PGE2, U46619, COX-1, COX-2 and TXB2 EIA Kit were purchased from Cayman Chemical Company (Ann Arbor, MI). Collagen (Collagenreagent Horm) was purchased from Nycomed Pharma GMBH (Marburg, Germany). Thrombin was purchased from Wako Pure Chemicals (Osaka, Japan). Arachidonic acid and ADP were purchased from Sigma-Aldrich (St. Louis, MO) or Cayman Chemical Company. All other chemicals used were of reagent grade or the highest quality available.
7
Anti-inflammatory Screening of Novel Compounds
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Silica gel (70–230 mesh, ASTM and 230–400 mesh) and Preparative Thin Layer Chromatography (TLC) were purchased from Merck. Deuterated chloroform (CDCl3), TPA, indomethacin, LPS from Escherichia coli serotype 055:B5, sodium nitrite (NaNO2), N-(1-naphtyl) ethylenediamine dihydrochloride and sulfanilamide were purchased from Sigma Aldrich. Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS) and Glutamine (GlutaMax) were from GIBCO, [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was from Promega Co. sPLA2, COX-1 and COX-2 ELISA kits were purchased from Cayman Chemical Co.
8
ELISA for Inflammation Biomarkers
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Enzyme-linked immunosorbent assay (ELISA) for PGE2 (Enzo Life Sciences, USA), Cox-1 (1:500, Cayman Chemicals, USA), TNF-α (PeproTech, Rocky Hill, NJ), and IL-1β (PeproTech, Rocky Hill, NJ) expression in retinas were performed using ELISA kits, respectively, according to manufacturer’s instructions as previously described by us [21 (link), 22 (link)]. The specificity of all the antibodies used for ELISA in mouse has been validated by the manufacturers.
9
Western Blot Analysis of Protein Expression
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Tissue samples were homogenized in a sucrose/imidazole buffer as described previously.62 Protein concentration was determined using the Quick Start Bradford Protein Assay (Biorad) using a serial dilution of BSA as standard. For western blotting, 10–30 μg of protein was separated by SDS‐PAGE in a 4%–12% Bis‐Tris gel (Invitrogen). By use of the XCell SureLock Mini‐Cell System (Invitrogen), proteins were transferred to an activated 0.45 μm pore‐size polyvinylidene difluoride membrane (Immobilon‐P Transfer Membrane; Millipore). Membranes were blocked with 5% nonfat dry milk in TBST (137 mM NaCl, 20 mM Tris at pH 7.6, 0.05% Tween 20) before incubation with primary antibodies. The antigen–antibody complex was visualized by horseradish peroxidase‐conjugated secondary antibodies (P0448, P0447, 1:2000; DAKO) using the enhanced chemiluminescence system (Amersham Pharmacia Biotech or PerkinElmer). Primary antibodies were COX‐1 (160 109, 1:1000; Cayman Chemicals), GAPDH (ab9585, 1:10 000; Abcam), α‐smooth muscle actin (α‐SMA) (ab5694, 1:10 000, Abcam), VEGF (ab46154, 1:10 000, Abcam), EP2 (101 750, 1:1000; Cayman Chemicals), and β‐tubulin (ABT171, 1:10 000; Sigma‐Aldrich). Non‐cropped images of membranes are available in supplement.
10
Sodium Selenite Modulates Cell Signaling
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Sodium selenite was obtained from LOBA Chemie (Mumbai, India), and Dulbecco's modi ed Eagle's medium (DMEM) and the antibiotics streptomycin and penicillin were obtained from HiMedia (Mumbai, India). Antibodies were used to detect EGR-1 (Santa-Cruz, Paso Robles, CA, USA), COX-1 (Cayman, Ann Arbor, MI, USA), Bcl-2 (Santa-Cruz, Paso Robles, CA, USA), Bax (Santa-Cruz, Paso Robles, CA, USA), caspase-3 (Sigma, St. Louis, MO, USA), caspase-8 (Cell Signaling Technology, Beverly, MA, USA), caspase-9 (Santa-Cruz, Paso Robles, CA, USA) and β-actin (Sigma, St. Louis, MO, USA). Anti-rabbit immunoglobulin (IgG) secondary antibody was obtained from Genei (Bengaluru, India). All other chemicals and reagents were of analytical grade. Milli-Q water (Millipore, Bengaluru, India) was used throughout the experiments.
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