Cox 2

To study COX-2 protein expression, RAW264.7 cells were treated for 6 hours with LPS or BLP (25 ng to 100 ng/ml). Lysates were prepared using Radio-Immunoprecipitation Assay (RIPA) buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for COX-2 (Cayman Chemicals, MI, USA) and GAPDH (Fitz Gerald Industries, MA, USA). The bands were visualized using SuperSignal West Pico ECL assay kits.

Western blots were performed as described [12 (link)] using antibodies against HuR (clone 3A2, Santa Cruz Biotechnology) at a dilution of 1:20,000 for 1 hr at RT and COX-2 (160126, Cayman Chemical) at a dilution of 1:1000 for 16 hours at 4°C. Caspase 3 cleavage was detected using rabbit polyclonal anti-Caspase 3 (9662, Cell Signaling) at a dilution of 1:1000 for 16 hr at 4°C. Membranes were stripped and re-probed using β-actin antibody (Clone C4; MP Biomedicals). Cytoplasmic lysates were obtained using NE-PER cytoplasmic extraction reagent (Thermo Scientific) using α-tubulin (322500, 1:20,000 dilution, Invitrogen) and Lamin A/C (2032S, 1:2,000 dilution, Cell Signaling) as cytoplasmic and nuclear loading controls, respectively. Detection and quantitation of blots were carried out as described [10 (link)].

Western blotting was carried out as previously described16 (link). Antibodies used in this study were listed as follows: CFTR (1:200, Cat#ACL-006, Alomone labs, Jerusalem, Israel), COX-2 (1:200, Cat#160106, Cayman chemical, Ann Arbor, USA), P-JNK (1:1000, Cat#9255, Cell Signaling Technology, Boston, USA), JNK (1:1000, Cat#9252, Cell Signaling Technology), P-Erk1/2 (1:1000, Cat#4370, Cell Signaling Technology), Erk1/2 (1:1000, Cat#4695, Cell Signaling Technology), P-IκBα (1:500, Cat#AB55066, Sangon Biotech), GAPDH (1:5000, Cat#KC-5G5, Kangcheng Biotech, Shanghai, China), Tubulin (1:500, Cat#10759-1-AP, Proteintech, Chicago, USA) and β-actin (1:1000, Cat#60008-1-lg, Proteintech).

Huh-7 cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well overnight and treated with indicated reagent at proper concentrations for three days. Cells were washed with cold phosphate-buffered saline (PBS) and lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM ethylene glycol bis(2-aminoethyl)tetraacetic acid (EGTA), 1 mM NaVO₃, 10 mM NaF, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 25 μg/mL aprotinin, and 25 μg/mL leupeptin) and stored at −20 °C. The protein concentration was determined by the Bradford method. Then, 10-μg protein samples were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% non-fat dried milk and incubated with specific antibodies against GAPDH (1:10000, Genetex, Irvine, CA, USA) and COX-2 (1:1000; Cayman Chemical, Ann Arbor, ML, USA). Antibodies were diluted in 5% milk containing Tris-buffered saline (TBS) and 0.5% Tween. The blotting signal was developed using an enhanced chemiluminescence (ECL) detection kit (PerkinElmer, Waltham, MA, USA) and was counted by the software Quantity One (Bio-Rad, Hercules, CA, USA).