2.6. Protein Extraction and Western Blotting Assay

Cells were collected and lysed in RIPA buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 µg/mL leupeptin (Cell Signaling Technology, Beverly, MA, USA). Then, the lysates were centrifuged at 12,000× g for 15 min at 4 °C. Supernatants were collected and total protein concentrations were determined using the DC protein assay (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Samples were separated by 10–15% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA) using a semidry electroblotting system (Bio-Rad, Hercules, CA, USA). Nitrocellulose membranes were incubated overnight at 4 °C with primary antibodies against the following proteins: COX-2, iNOS, NF-κB p65, phosphorylated NF-κB p65 (p-p65), IκBα, and p-IκBα. After washing off the primary antibody, the blots were incubated for 2 h with mouse or rabbit secondary antibodies (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive protein bands were detected using an enhanced chemiluminescence (ECL) detection kit and captured with the CAS-400SM Davinch-Chemi chemiluminescence imaging system.