A12621

The tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor. The protein samples were mixed with 5 × dual color protein loading buffer (Fudebio, Hangzhou, China) and boiled at 98 °C for 10 min. Equal amounts of protein (30 μg/lane) were loaded on a SDS-PAGE gel at a constant voltage and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After being blocked and incubated with the primary and secondary antibodies, the bands were visualized using a chemiluminescence image analysis system (Tanon, Shanghai, China) with FDbioFemto ECL (Fudebio, Hangzhou, China). The band intensities were quantified using Gel-Pro Analyzer software (Media Cybernetics, Maryland, USA). The primary antibodies were included the following: synaptosomal associated proteins 25 (SNAP-25; Abcam, ab109105), postsynaptic density proteins 95 (PSD-95; CST, 3450), Ibα1 (Abcam, ab178846), TNF-α (Abcam, ab215188), IL-6 (Abcam, ab233706), NLRP3 (Abcam, ab263899), IL-18(Abcam, ab243091), IL-1β (Abcam, ab254360), caspase-1 (Abcam, ab207802), TLR4 (Abcam, ab22048), myeloid differentiation factor 88 (MyD88; Abcam, ab133739), NF-κB p65 (Abcam, ab32536), GAPDH (ABclonal, AC033), ZO-1 (ABclonal, A11417), occludin (ABclonal, A12621) and β-actin (ABclonal, AC026).