Ecl system

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.

Cells were rinsed with PBS and lysed in RIPA buffer containing a protease inhibitor cocktail for about 30 min. After that, the lysate was collected and centrifuged for 15 min at 12000 rpm and the protein in the supernatant was detected by BCA protein assay kit (Servicebio, Wuhan, China). The cell lysates mixed with protein loading buffer and loaded in SDS-polyacrylamide gels then transferred to PVDF membranes (Millipore, Billerica, MA). Then, membranes were blocked and incubated with specific primary antibodies. Before visualizing by ECL system (biosharp, Hefei, China), membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies.