Cocktail protease inhibitors and phosphatase inhibitors

After NLEE or NLWE pre-treatment, cells were stimulated with LPS for either 15 min or 18 h and then lysed in RIPA lysis buffer containing cocktail protease inhibitors and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA). Cell debris was removed by centrifugation for 30 min at 10,000 g at 4℃. Protein concentrations were determined using a Bradford Assay kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions. Lysates were boiled in sample buffer for 5 min. Equivalent amounts of sample protein were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature, and then probed with antibodies against iNOS, COX-2, phosphorylated inhibitor of NF-κB (Iκ-B)-α, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were washed and then exposed to horseradish peroxidase-conjugated secondary antibodies (1:4000 dilutions). Immunoreactive bands were detected by enhanced chemiluminescence solution (Animal Genetics, Seoul, South Korea) and exposed to X-ray film.

After RCM treatments, cells were stimulated with LPS and lysated in RIPA lysis buffer containing cocktail protease inhibitors and phosphatase inhibitors (Sigma-Aldrich, St. Louis, USA). Immunoblot assay was carried out as described previously [27 (link)] with antibodies against iNOS, COX-2, p-IκB, ERK 1/2, p-ERK 1/2, p38, p-p38, JNK, p-JNK, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA).