*4.5. Protein Extraction*

The cells were washed three times with phosphate-buffered saline (PBS) chilled to 4 ◦C. Whole-cell proteins were extracted with M-PER Mammalian Protein Extraction Reagent (78503, Thermo Fisher Scientific, Waltham, MA, USA), containing protease and phosphatase inhibitor (Roche, Mannheim, Germany), at 4 ◦C for 30 min. Then, the samples were centrifuged at 14,000× *g* for 10 min, and the supernatant was transferred to a new tube for analysis. Nuclear proteins were extracted in accordance with the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Biotechnology, Shanghai, China) for detection of the activity of NF-κB.
