Anti cox 2 antibody

For western blotting, cell lysate was prepared with RIPA buffer containing NaCl 150 mM, Tris-HCl (pH 8.0) 50 mM, 1% Triton X-100, 0.5% Na deoxycholate, 0.1% SDS, EDTA 1 mM, EGTA 1 mM, Na₃VO₄ 1 mM, β-glycerophosphate 1 mM, and the protease inhibitor cocktail from Roche Applied Sciences for 5 min on ice, followed by centrifugation at 12,000× g for 5 min at 4 °C to remove debris. The transferred nitrocellulose membrane was pre-incubated with 5% BSA in PBS+0.05% Tween 20 (PBST) for 30 min and then incubated with anti-β-actin antibody (AC-74, Sigma-Aldrich) at 1:2000, anti-iNOS antibody (Cat# 15323, Abcam, Cambridge, UK) at 1:1000, anti-FAAH antibody (Cat# 54615, Abcam) at 1:1000, or anti-COX-2 antibody (Cat# 160106, Cayman Chemical) at 1:500 in PBST at 4 °C overnight. The membrane was reacted with a secondary antibody conjugated with horse radish peroxidase (Bio-Rad) at 1:2500 for 1.5 h, followed by visualization with ECL reagent (Thermo Scientific), using an imaging system (ChemiDoc, Bio-Rad) with chemiluminescent mode. Band intensity was quantified with NIH ImageJ software.

Dulbecco’s modified Eagle’s medium (DMEM) and lipopolysaccharide (LPS) were obtained from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Shiga toxin-2 was obtained from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum (FBS) and interleukin-1 receptor-associated kinase (IRAK)-1/4 inhibitor were obtained from Invitrogen Corporation (Carlsbad, CA, USA) and Calbiochem (Billerica, MA, USA), respectively. Anti-phospho-specific nuclear factor-κB (NF-κB) p65 (Ser536), anti-NF-κB p65, anti-phospho-specific extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), anti-ERK, anti-β-actin, horseradish peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-COX2 antibody was obtained from Cayman Chemical Company (Ann Arbor, MI, USA). An antibody against AQP4 was purchased from Millipore (Billerica, MA, USA).

Western blotting was performed as previously described. Anti-COX-2 antibody was obtained from Cayman, Ann Arbor, MI. Anti-GAPDH and anti-lamin B antibodies were obtained from GeneTex. Anti-phosphorylated IκBα, IKKα/β, p38, JNK, ERK and anti-total iNOS, IκBα, IKKα, IKKβ, p65, p38, JNK, ERK antibodies were obtained from Cell Signaling (Beverly, MA, USA).

The standard procedure of Western blotting was performed as described previously (Lee et al., 2011 (link)). The membranes were probed with monoclonal antibodies specific for HCV NS5B (1:5000; Abcam, Cambridge, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000; GeneTex, Irvine, CA, USA), anti-COX-2 antibody (1:1000; Cayman, MI, USA), anti-MAPK (phosphorylated and unphosphorylated forms of ERK1/2, p38, and JNK), anti-IKKα, anti-phospho-IKKα/β (Ser176/180), anti-NF-κB, anti-IκB-α, anti-phospho-IκB-α (Ser32) (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), or anti-C-Myc antibody (1:1000; GeneTex, Irvine, CA, USA). The ECL detection kit was used for the signal detection (PerkinElmer, Shelton, CT, USA).

Western blotting was carried out as described previously [27 (link)]. In brief, an equal volume of cellular lysate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to a PVDF membrane. The membranes were probed with antibodies specific to DENV NS2B (1:5000; Abcam, Cambridge, MA, USA), anti-GAPDH (1:10,000; GeneTex, Irvine, CA, USA), anti-COX-2 antibody (1:1000; Cayman Chemical, Ann Arbor, MI, USA), anti-MAPK (phosphorylated and unphosphorylated forms of ERK1/2, p38, and JNK), and anti-C-Myc antibody (1:1000; GeneTex).