Occludin 27260 1 ap

The high-fat diet (HFD, D12492) chow was purchased from Beijing Huafukang Bioscience; standard rat chow (SRC) was offered by the experimental animal center of Wuhan University Renmin Hospital. Rat ELISA kits of interleukin- (IL-) 1β (E-EL-R0012c) and IL-6 (E-EL-R0015c) were purchased from Elabscience (Wuhan, China). Diamine oxidase (DAO) assay kit (A088-1) and endotoxin assay kit (E039-1-1), reduced form of glutathione (GSH) assay kit (A006-2-1) and maleic dialdehyde (MDA) assay kit (A003-1-2), and superoxide dismutase (SOD) assay kit (A001-2-2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Zonula occluden-1 (Zo-1) (21773-1-AP) and occludin (27260-1-AP) antibodies were purchased from Proteintech (Wuhan, China); RIP3 (ab56164) and TNF-α (ab6671) antibodies were purchased from Abcam (Cambridge, UK); TLR4 (GB11519), NF-κB P65 (GB11142), and β-actin (GB11001) antibodies were purchased from Servicebio (Wuhan, China). TAK242 (HY-11109), an inhibitor of TLR4, was purchased from MCE (Shanghai, China).

The tissues were lysed using RIPA lysis buffer (Beyotime Tech, Shanghai, China) with 1% PMSF (Beyotime Tech, Shanghai, China) and phosphatase inhibitor cocktail (BosterBio, Nanjing, China) to extract total protein. The concentration of proteins was determined by using a BCA assay (KeyGen Biotech, Nanjing, China). Tissue protein (30 μg/lane) was electrophoresed through SDS-PAGE at 90 V for 15 min and 120 V for 1 h, and then, electronically transferred to PVDF membranes at 260 mA for 1 h. After blocking with 5% de-fat milk for 2 h, the blots were incubated at 4 °C overnight with primary antibodies: β-actin (13E5) (purchased from Cell Signaling Technology (Boston, USA); ZO-1(21773-1-AP), occludin (27260-1-Ap), caspase-3 (66470-2-Ig), and JAK1 (66466-1-Ig) (purchased from Proteintech (Wuhan, China)); NF-κB p65 (ab32536), STAT3 (ab68153), and Nrf2 (ab76036) (purchased from Abcam (ab205718 and ab205719, Cambridge, UK)). After washing with TBST buffer three times, the HRP-conjugated secondary antibodies (Abcam, Cambridge, UK) were applied for 2 h at room temperature. Washing was performed again, and then the chemiluminescence signals were detected by using an ECL system (Biosharp, Hefei, China) and visualized by using a Chemiluminescence imager (Bio-Rad, California, USA). Finally, the images were analyzed using the Image Lab software. All expression levels were normalized to β-actin.