Primary antibody against phosphorylated p65

The total protein of THP-1-derived macrophages was extracted and protein concentration was measured in accordance with the above methods and steps. Protein samples (20 μg) were separated in 10% SDS-PAGE and then electrically transferred onto the PVDF membrane. The membrane was blocked in skim milk for 1 hour and incubated with primary antibody against phosphorylated p65 (Cell Signal Technology, Boston, MA, USA) at 4 °C for overnight, and then oscillatorily incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour. Immunoreactions were visualized by ECL and X-ray film (Eastman Kodak Company, Rochester, NY, USA) exposure. After exposure, the membrane was stripped with stripping solution for 30 minutes, blocked by milk, and incubated with primary antibody against NF-κB p65 at 4 °C overnight. It was then incubated with a secondary antibody and immunoreactions were visualized after exposure.

The tumor tissues were fixed with 4% paraformaldehyde and subsequently sectioned into 5-µm-thick slides. These slides were then subjected to standard H&E staining procedures. For IHC analysis, the slides were incubated overnight at 4 °C with a primary antibody against phosphorylated p65 (Cell Signaling Technology, Danvers, MA, USA). Following this, the slides were incubated with HRP-conjugated streptavidin for 1 h at room temperature. Visualization was achieved using the DAB chromogen (Zhongshan Golden Bridge, Beijing, China). Images were captured at 100 × and 400 × magnifications using a microscope (Zeiss, Oberkochen, Germany).