Phosphate-buffered saline (PBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Corning, Inc. (New York, NY, USA)., fetal bovine serum (FBS) were obtained from Gibco (Grand Island, CA, USA). Penicillin-Streptomycin was Thermo fisher (Waltham, MA, USA). Lipopolysaccharide (LPS, Escherichia coli 055:B5) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against rabbit-anti COX-2, NF-κB p65, ERK, phospho-extracellular signal-regulated kinase (p-ERK), p38, p-p38, JNK, phospho-Jun N-terminal (p-JNK), phosphoinositide 3-kinase (PI3K), p-AKT, AKT, and Lamin B1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-iNOS was purchased from from R&D Systems Inc.(Minneapolis, MN, USA). IL-1β and TNF-α were purchased from Cruise Biotechnology (Santa Cruz, CA, USA). Mouse anti-β-actin and goat anti-rabbit IgG HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Rabbit anti cox 2
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-COX-2 is a primary antibody that specifically recognizes the Cyclooxygenase-2 (COX-2) protein. COX-2 is an enzyme involved in the production of prostaglandins, which play a role in inflammatory responses. This antibody can be used for the detection and analysis of COX-2 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Santa Cruz Biotechnology (5 mentions)
Rabbit anti inos, by Cell Signaling Technology (4 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Pierce s nitrosylation western blot kit, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (2 mentions)
Cary uv vis spectrophotometer, by Agilent Technologies (2 mentions)
Rabbit anti p p65, by Cell Signaling Technology (2 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti p akt, by Cell Signaling Technology (2 mentions)
Rabbit anti erk, by Cell Signaling Technology (2 mentions)
Celecoxib, by Merck Group (2 mentions)
Rabbit anti actin, by Santa Cruz Biotechnology (2 mentions)
Celebrex, by Merck Group (2 mentions)
Rabbit polyclonal anti akt and anti pakt, by Cell Signaling Technology (2 mentions)
Pseudomonas aeruginosa lps, by Merck Group (2 mentions)
Flagellin, by Novus Biologicals (2 mentions)
Ly94002, by Cell Signaling Technology (2 mentions)
Rabbit anti ho 1, by Abcam (2 mentions)
Ibuprofen, by Merck Group (2 mentions)
20 protocols using rabbit anti cox 2
1
Inflammatory Signaling Pathway Activation Protocol
2
COX-2 S-nitrosylation and n-3 DPA Metabolism
3
Western Blot Analysis of Cellular Senescence
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Snap frozen tissues from Ercc1−/Δ and WT mice were lysed in RIPA lysis and extraction buffer (Thermo Fisher). Protein concentration was determined using BCA protein assay kit (Thermo Fisher). Equal amounts of protein were loaded onto SDS‐PAGE polyacrylamide gels then transferred to 0.2 µm pore size nitrocellulose membranes (Bio‐Rad). Membranes were blocked with 5% BSA in PBS‐Tween for 1 h at room temperature then incubated with primary antibodies overnight at 4℃. Membranes were then probed with secondary antibodies at room temperature. Protein expression was measured by fluorescence using iBright™ FL1000 Imaging System. The density of each blot was quantified by using ImageJ (NIH) normalized to GAPDH. The following primary antibodies were used in this study: rabbit anti‐GAPDH (Cell Signaling Technology, 2118), rabbit anti‐p16 (Santa Cruz, sc‐1207), rabbit anti‐p21 (Abcam, ab7960), rabbit anti‐p‐p65 (Cell Signaling Technology, 3033), mouse anti‐p‐p65 (Cell Signaling Technology, 6956), rabbit anti‐Pai‐1 (Santa Cruz, sc‐8979), rabbit anti‐COX2 (Cell Signaling Technology, 12282). The following secondary antibodies were used in this study: goat anti‐rabbit IgG (H+L) Alexa Fluor Plus 488 (Thermo Fisher, A32731), goat anti‐mouse IgG (H+L) Alexa Fluor 633 (Thermo Fisher, A21052).
4
Antibody Characterization for Cellular Analysis
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The following antibodies were used: mouse anti-F4/80 (Santa Cruz), rabbit anti-iNOS (Cell Signaling Technology), mouse anti-CD80 (Invitrogen Antibodies), rabbit anti-CD206 (Abcam), rabbit anti-SHP2 (Cell Signaling Technology), rabbit anti-COL2 (Abcam), rabbit anti-COL10 (Abcam), rabbit anti-MMP3 (Proteintech), rabbit anti-COX2 (Cell Signaling Technology), rabbit anti-GAPDH (Cell Signaling Technology), rabbit anti-p-P65 (Cell Signaling Technology), rabbit anti-P65 (Cell Signaling Technology), rabbit anti-β-actin (Cell Signaling Technology), rabbit anti-histone H3 (Cell Signaling Technology), rabbit anti-p-AKT (Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), and rabbit anti-p-IKKα/β (Cell Signaling Technology).
5
COX-2 S-nitrosylation and n-3 DPA Metabolism
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Human recombinant COX-2 (10 units; Cayman Chemicals; in 0.1M Tris-HCl, pH 8.0, 20μM porcine hematin, 0.67mM phenol as in 33 (link)) was incubated with the indicated concentrations of AA or n-3 DPA (1h, RT) and absorbance at 235nM was investigated at 1 min intervals using a Cary UV-Vis Spectrophotometer (Agilent).
In designated experiments, n-3 DPA was incubated with hrCOX2, hrCOX-2 that was previously incubated with S-nitrosoglutathione (30 min RT), prepared by incubating 100mM glutathione with 100 mM sodium nitrite in 200mM hydrochloric acid at RT as in 34 (link) or S-nitrosoglutathione. After 20 min the incubations were quenched with methanol and 13-HDPA levels were assessed using LC-MS-MS-based LM metabololipidomics.
COX-2 S-nitrosylation was assessed using a Pierce Western Blot S-nitrosylation Kit (Thermo Fisher) following manufacturer’s instructions, and total COX-2 levels were determined using a rabbit anti-COX-2 (Cell Signaling) antibody and a goat anti-rabbit-HRP conjugated antibody (eBioscience).
In designated experiments, n-3 DPA was incubated with hrCOX2, hrCOX-2 that was previously incubated with S-nitrosoglutathione (30 min RT), prepared by incubating 100mM glutathione with 100 mM sodium nitrite in 200mM hydrochloric acid at RT as in 34 (link) or S-nitrosoglutathione. After 20 min the incubations were quenched with methanol and 13-HDPA levels were assessed using LC-MS-MS-based LM metabololipidomics.
COX-2 S-nitrosylation was assessed using a Pierce Western Blot S-nitrosylation Kit (Thermo Fisher) following manufacturer’s instructions, and total COX-2 levels were determined using a rabbit anti-COX-2 (Cell Signaling) antibody and a goat anti-rabbit-HRP conjugated antibody (eBioscience).
6
Inflammatory Pathway Modulation Protocols
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Amlexanox was purchased from Tokyo Chemical Industry (Tokyo, Japan). STAT3 activation inhibitor SPI was purchased from BioVision (Milpitas, CA, USA). LPS from Escherichia coli O111:B4 was obtained from Sigma–Aldrich (St. Louis, MO, USA). The following primary antibodies, which were diluted at 1:1000 ratios in EveryBlot Blocking Buffer (Bio-Rad Laboratories), were used for Western blotting (WB): rabbit anti-COX2 (Cell Signaling Technology), mouse anti-iNOS (Cell Signaling Technology), rabbit anti-p-AKT (Ser473, Cell Signaling Technology), rabbit anti-AKT (Cell Signaling Technology), rabbit anti- IκBα (Cell Signaling Technology), rabbit anti-p- IκBα (Ser32, Cell Signaling Technology), rabbit anti-IKKε (Cell Signaling Technology), rabbit anti-ERK (Cell Signaling Technology), rabbit anti-p-ERK (Thr202/Tyr204, Cell Signaling Technology), rabbit anti-STAT3 (Cell Signaling Technology), rabbit anti-p-STAT3 (Tyr705, Cell Signaling Technology), rabbit anti-NF-κB p65 (Cell Signaling Technology), rabbit anti-p-NF-κB p65 (Ser536, Cell Signaling Technology), rabbit anti-JNK, rabbit anti-p-JNK (Thr183/Tyr185, Cell Signaling Technology), rabbit anti-p38 (Cell Signaling Technology), and rabbit anti-p-p38 (Thr180/Tyr182, Cell Signaling Technology), and mouse anti-β-actin (Santa Cruz Biotechnology). Other chemicals for Western blotting were obtained from Bio-Rad Laboratories.
7
Western Blot Quantification Protocol
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Ten percent SDS-PAGE was used to separate protein samples (about 80 μg protein), which were then transferred to PVDF membranes (Millipore, Billerica, MA, USA). PVDF membranes were then blocked by 5% BSA for 1 h at 37°C. PVDF membranes were cut off in accordance with the molecular weight, and incubated with different primary antibodies (rabbit anti-GAPDH, 1:1000 dilution; mouse anti-RAGE, 1:800; rabbit anti-p-NF-κB P65, 1:800; mouse anti-GFAP, 1:1000; rabbit anti-Iba-1, 1:800; mouse anti-p-JNK, 1:800; rabbit anti-PARP, 1:1000 from Santa Cruz Biotechnology; rabbit anti-COX2, 1:1000; rabbit anti-iNOS, 1:1000 from Cell Signaling Technology) at 4°C overnight. On the second day, PVDF membranes were incubated with HRP-conjugated secondary antibody (anti-mouse and anti-rabbit, 1:2000) for 2 h at 37°C. Protein bands were detected by an ECL western blotting kit using a ChemiDoc-It™ imaging system (UVP, Upland, CA, USA). GAPDH was selected as a control. The gray value was analyzed by Gel-pro 32 (Media Cybernetics, Rockville, MD, USA).
8
Anti-inflammatory Signaling Pathway Modulation
9
Anti-inflammatory Signaling Pathway Modulation
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The following antibodies were used: rabbit anti-HO-1 (1:2000 for western blot, Abcam); rabbit anti-COX-2 (1:1000, Cell Signaling Technology); rabbit polyclonal anti-AKT and anti-pAKT (1:1000, Cell Signaling Technology); rabbit anti-actin (1:5000, Santa Cruz); anti-rabbit-HRP (1:2000, Santa Cruz). Pseudomonas aeruginosa (PA) LPS (Sigma-Aldrich) was prepared in PBS at 100X stock solution and used at a concentration of 100 ng/mL. Flagellin (Imgenex, San Diego CA) was used at a concentration of 100 ng/ml). CFTR inhibitor CFTRinh172, a kind gift from Dr. Alan Verkman)32 (link), was freshly prepared in DMSO and used at concentration of 20μM. The PI3K/AKT inhibitor LY94002 (Cell Signaling Technology) was prepared in DMSO and used at a concentration of 20μM. The AKT Activator II, SC79 (Merck Millipore) was dissolved in DMSO and used at the final concentrations indicated. Celecoxib (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 20 mM) and used at a final concentration of 25μM. For the in vivo study we used the FDA approved branded Celecoxib (Celebrex) at concentration of 25mg/kg/mouse/day. Ibuprofen (Sigma-Aldrich, St Louis MO) was dissolved in DMSO (stock solution 50 mM) and used at a final concentration of 25μM or 100μM. For the in vivo study we used Walgreens Children’s Ibuprofen Suspension (20mg/ml) at a final concentration of 50 mg/kg/mouse/day.
10
Western Blot Analysis of Inflammatory Markers
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Cells were plated and treated with each RSL chloroform fraction for 1 h and LPS (1 μg/mL; Sigma) was added. After 18 h incubation, cells were lysed with ice-cold lysis buffer with protease inhibitors (Sigma). The protein concentration was measured with the Bradford protein assay reagent (Bio-Rad Laboratories). Equal amounts of proteins were loaded to each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blocked in 5% non-fat milk. Samples were probed with the following primary antibodies: mouse anti-iNOS (Abcam, Cambridge, MA, USA), rabbit anti-COX-2 (Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and mouse anti-β-actin (Santa Cruz Biotechnology) antibodies. The secondary antibodies used were either goat anti-mouse IgG or anti-rabbit IgG-peroxidase conjugates (Amersham Biosciences, Uppsala, Sweden). The membranes were incubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology).
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