Detection of both NF-κB and p38 MAPK activation by western blot

The tissue samples (50–100 mg) were homogenized in 1 mL of NP-40 lysis buffer supplemented with protease and phosphatase inhibitors and centrifuged at 12,000 × g for 15 min at 4 °C. Tissue proteins were separated by 12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the nitrocellulose membranes. After blocking with 3 % BSA in Tris-buffered saline including 0.1 % Tween-20 buffer, membranes were incubated with primary antibodies against NF-κBp65 (Cell Signaling), phospho-NF-κBp65 (Cell Signaling), p38 MAPK (Cell Signaling), phospho-p38 MAPK (Cell Signaling) or β-actin (Sigma-Aldrich). Membranes were then washed and incubated with appropriate secondary antibodies. Proteins were visualized using the ECL reagent according to the manufacturer’s instructions. One sample from each treatment was run in duplicate in a gel and 12 samples from 4 treatments (n = 3) were run in 3 gels at one time to minimize the variations of gel to gel. Densitometric analysis was performed by Gel-Pro analyzer and normalized to β-actin.