2.4. Western Blot Analysis

HPDLCs were pretreated with nobiletin (25, 50, or 100 μM) for 1 hour before the stimulation with TNF (10 ng/ml) for 15, 30, or 60 min (analysis for the activation of signal transduction pathway) or 24 hours (analysis for the PTGS2, HMOX1, and NFE2L2 expression). Then, the cell supernatant was discarded and washed twice with cold phosphate-buffered saline (PBS). HPDLCs were lysed with cell lysis buffer (Cell Signaling Technology) including protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Debris was removed by centrifugation at 15000 rpm for 10 min at 4°C, and the supernatants were collected. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bradford, USA). The membranes were blocked with 1% skim milk for 1 hour at room temperature. Then, the membranes were incubated with the following primary antibodies at 4°C overnight: phospho-MAPK14 (1 : 1000), phospho-MAPK1/MAPK3 (1 : 2000), phospho-MAPK8 (1 : 1000), phospho-IKK-α/β(1 : 1000), phospho-NF-κB p65 (1 : 1000), phosph-AKT1 (1 : 2000), MAPK14 (1 : 1000), MAPK1/MAPK3 (1 : 2000), MAPK8 (1 : 1000), IKK-α(1 : 1000), NF-κB p65 (1 : 1000), AKT1 (1 : 2000), PTGS2 (1 : 1000), HMOX1 (1 : 1000), NFE2L2 (1 : 1000), or GAPDH (1 : 8000). The membranes were washed with Tris-buffered saline including 0.1% Tween 20 (TBS-T) 3 times and exposed to horseradish peroxidase-conjugated secondary antibodies (Sigma) for 1 hour at room temperature. The membranes were washed with TBS-T 3 times, and the protein bands were visualized by the Enhanced Chemiluminescence (ECL) Plus Western blotting detection system (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions. The experiments were repeated three times. The densities of bands of Western blot analysis were determined using ImageJ software (NIH, Bethesda, MD, USA).