2.9. Western blot assay for the expressions of the NF-κB signaling pathway and EMT-related proteins in tumor tissues

A sample of 20 g of tumor tissue was weighed into a metal lysis medium S tube, added with 300 μL of Radio-Immunoprecipitation Assay strong lysate, and ground with a tissue sample grind. The lysate was pipetted into a 1.5-mL centrifuge tube and lysed at 4 ℃ for 30 min with shaking. The lysate was centrifuged at 13 000 rpm for 10 min at 4 ℃, the supernatant was separated and placed in a 1.5-mL centrifuge tube, and stored at −80 ℃. The protein concentration was determined by a BCA protein quantitative kit, transferred to a polyvinylidene fluoride (PVDF) membrane, blocked with 5% skim milk powder at room temperature for 2 h, washed in a 1 × Tris Buffered Saline with Tween-20 (TBST) buffer solution on a shaker for 10 min three times, and incubated overnight with primary antibody at 4 ℃. The PVDF membrane was immersed in 1 × TBST buffer, washed three times on a shaker for 10 min each time, and incubated with the secondary antibody for 2 h on a shaker at room temperature. The PVDF membrane was immersed in 1 × TBST buffer, washed three times on a shaker for 10 min each, placed on plastic wrap, and an appropriate amount of electrochemiluminescence was applied to the surface of the membrane and transferred to a gel imaging analyzer for exposure. The intensities of the Western blot bands were measured using ImageJ software (V1.6.0, W.S. Rasband, Bethesda, MD, USA).