Protein Extraction and Quantification Protocol

The protein extraction and quantification were applied according to our previous literature^{13 (link)}. The protein concentration was determined by the bicinchoninic acid (Beyotime Institute of Biotechnology, China) method. The protein samples were separated by SDS-PAGE (Beyotime Institute of Biotechnology, China) electrophoresis. The film was transferred under the condition of voltage 100 V, current 120 mA, 90–120 min. It was then sealed in a sealant (5% skim milk) for 2 h. TTBS was washed off the sealing fluid, and the primary antibody was diluted with primary antibody diluen: ZAK (1:500; abcam, USA), p-NFκB-p65 (1:1000; Thermo Scientific, Beijing, China), NFκB-p65 (1 µg/ml; Thermo Scientific, Beijing, China), ZO-1 (1:300; Thermo Scientific, Beijing, China), occludin (1:200; Thermo Scientific, Beijing, China), and claudin-5 (1:500; Thermo Scientific, Beijing, China) in a certain proportion and sealed with a film at 4 °C overnight. After washing TTBS for three times, add the corresponding secondary antibody and incubate at room temperature for 2 h. After TTBS washing for three times, ECL (Beyotime Institute of Biotechnology, China) was used to glow, take photos, and quantity one software was used for quantitative analysis.