Pink1

Protein isolated from IPEC-J2 cells was separated by SDS gel electrophoresis and transferred to PVDF membranes. Then, membranes were incubated with tight junction primary antibodies (Claudin-1, Claudin-3, ZO-1, N-cad and Occludin) (Biosynthesis Biotechnology Inc., Beijing, China), mitophagy primary antibodies (LC3, P62, Beclin1, Parkin and PINK1) (ABclonal, Wuhan, China), a housekeeping antibody (β-actin) and a secondary antibody (ABclonal, Wuhan, China), respectively. Finally, the bands were quantified using a Tanon-5200 (Tanon, Shanghai, China) [29 (link),30 (link)].

Protein samples (60–100 μg/well) were attached to nitrocellulose membranes (PALL, New York, USA) by electrophoresis and transfer membrane. Sealed with 5% skim milk, the protein binds to the primary antibodies for p21 (1:500, #sc‐817, Santa Cruz Biotechnology, CA, USA), p53 (1:1000, #25241, Cell Signaling Technology, MA, USA), LC3 (1:500, #12741, Cell Signaling Technology, MA, USA), p62 (1:1000, #5114, Cell Signaling Technology, MA, USA), Parkin (1:500, #sc‐133167, Santa Cruz Biotechnology, CA, USA), PINK1 (1:1000, #A7131, ABclonal, Wuhan, China), BNIP3 (1:1000, #A5683, ABclonal, Wuhan, China), VDAC (1:1000, #4661S, Cell Signaling Technology, MA, USA), GAPDH (1:2000, # TA802519, Clone OTI2D9, OriGene, MD, USA) on a shaker at 4°C overnight. Odyssey Infrared Imaging System (LI‐COR, Lincoln, NE, USA) was used to observe and quantify the experimental results of western blot.

The following reagents were used: DMEM, FBS, penicillin/streptomycin (Meilunbio, Dalian, China), cTnI Elisa kits (Sangon Biotech, Shanghai, China), TNF-α, IL-1β, and IL-6 Elisa kits (MyBioSource, San Diego, United States), MDA kits, SOD kits, and NADH kits (Solarbio, Beijing, China), CCK-8 kits (Beyotime Biotechnology, Shanghai, China), ROS fluorimetric kits, JC-1 fluorescent dye, protein extraction kits, and BCA kits (Meilunbio, Dalian, China). IL-1 (1:100), PINK1(1:1,000), Parkin (1:1,000), AMPK(1:1,000), Akt (1:1,000), P13K(1:1,000), Tom20 (1:1,000), β-tubulin (1:1,000), mTOR (1:1,000), and an HRP-conjugated GAPDH (1:1,000) antibody were obtained from Abclonal Biotechnology (Wuhan, China). The RIPA lysis buffer and loading buffer were purchased from Meilunbio (Dalian, China). PVDF membranes were obtained from Merck (New Jersey, United States). DMSO was purchased from Macklin (Shanghai, China), and doxorubicin, breviscapine, and dexrazoxane were purchased from Widely (Wuhan, China). The Mdivi-1 inhibitor was purchased from Selleck Chemicals (Houston, United States).