Adi spa 810 f

The expression levels of proteins were performed using western blot. Tissue extracts were prepared using RIPA buffer. After electrophoresis, proteins were transferred onto PVDF membranes. Then the membrane was blocked for 1 h at room temperature with 5% milk solution, and incubated with primary antibodies at 4°C overnight. The following antibodies were used in this study: NF-κB (6956S, Cell Signaling Technology), pNF-κB (3033, Cell Signaling Technology), Mx1 (ab79609, Abcam), IFN-α (ab230993, Abcam), HSP70 (ADI-SPA-810-F, Enzo Life Sciences), I-FABP (sc-16063, Santa Cruz Biotechnology), Caspase-3 (9661S, Cell Signaling Technology), Villin (sc-7672, Santa Cruz Biotechnology), AQP4 (ab46182, Abcam), AQP3 (ab125219, Abcam), Claudin (RF217968, Invitrogen), and Occludin (TC259714, Invitrogen), β-actin (PA1-46296, Invitrogen). Subsequently, appropriate HRP-conjugated secondary antibodies were used followed by incubation at room temperature for 1 h. Blots were visualized using a chemiluminescence kit (Amersham Biosciences, Uppsala, Sweden) and image forming system (Alpha Innotech, New York, NY, USA).

Mouse monoclonal anti-Hsp40 (ab78437; 1:5000), anti-Hsp90 (ab13492; 1:5000), anti-Hsp27 (ab2790 1:2500), anti-αB-c (ab13496; 1:5000), anti-mCherry (ab125096; 1:2000) and IgG1-isotype control (ab91353) primary antibodies, and goat anti-mouse IgG DyLight 488 conjugated seconday antibodies (ab96871) were obtained from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-Hsp70 primary antibody (ADI-SPA-810-F; 1:1000) was from Enzo Life Sciences (Farmingdale, NY, USA). Mouse monoclonal anti-α-tubulin primary antibody (T8203; 1:5000) and rabbit polyclonal anti-mouse IgG horse-radish peroxidase conjugated secondary antibody (SAB3701084; 1:5000) were obtained from Sigma Aldrich. Dilutions used for immunoblotting are included in parentheses.

The protein of jejunum was extracted by a whole protein extraction kit (KeyGEN, Jiangsu, China), and the protein concentration was quantified by a BCA kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE gels, followed by transferring onto PVDF membranes (Millipore, Billerica, MA, USA). The membrane bands were incubated with primary antibodies overnight at 4°C, and then incubated with secondary antibodies for 2 h at the room temperature. Finally, the grayscale value of protein band was determined by using an imaging system (Alpha Innotech FluorChem FC2, CA, USA). Antibodies used in the present study were as follows: MX1 (ab222856, Abcam, 1:1000), ISG15 (ab233071, Abcam, 1:1000), HSP70 (ADI-SPA-810-F, Enzo Life Sciences, 1:1000), IRF7 (QC8422, Sigma, 1:1000), p-IRF7 (PA5-114591, Invitrogen, 1:2000), JAK2 (#3230, Cell Signaling Technology, 1:2000), p-JAK2 (#3776, Cell Signaling Technology, 1:2000), STAT3 (#30835, Cell Signaling Technology, 1:1000), p-STAT3 (#9145, Cell Signaling Technology, 1:1000), Occludin (TC259714, Invitrogen, 1:2000), ZO-1 (61-7300, Invitrogen, 1:2000), Claudin-1 (RF217968, Invitrogen, 1:2000), E-cadherin (PA5-142828, Invitrogen, 1:2000), and β-actin (PA1–46296, Invitrogen, 1:4000).