2.7. Western Blot Analysis

Western blot analysis was carried out using antibodies targeting NF-κB p65, IκBα, iNOS (Santa Cruz Biotechnology), JNK, phospho-JNK, p38, phospho-p38, ERK, and phospho-ERK (Cell Signaling Technology, Beverly, MA, USA). To obtain proteins, the treated cells were centrifuged, washed with PBS three times, and lysed in RIPA lysis buffer including propidium iodide (UpstateBiotechnology, Lake Placid, NY, USA). After removing the cellular debris, the supernatant was obtained and stored at −20 °C until use. The protein concentration in lysates was measured with the Bradford method using standard controls.

Whole cell lysates were mixed with 4X LDS sample buffer (Invitrogen), boiled for 5 min, and the samples containing 40 μg of protein were subjected to 10% SDS-PAGE. The proteins were then transferred to Hybond-ECL nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% non-fat milk in TBST buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h. The membranes were incubated with the primary antibodies (1:2000) diluted in blocking buffer for 1 h at room temperature, and then incubated with the secondary horseradish peroxidase-conjugated antibodies (1:10,000). The target proteins were detected on X-ray film using ECL Advanced Western Blotting Detection Kit (GE Healthcare). GAPDH was used as a loading control.