Ecl chemiluminescence kit

After 2 h of exposure to DON, IPEC-J2 cells were collected and lysed in cell lysis buffer (Beyotime, Haimen, China). Protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Proteins were separated by electrophoresis and transferred to PVDF membranes. Anti-phospho-p38 (1:1000), anti-p38 (1:1000), anti-phospho-JNK (1:1000), anti-JNK (1:1000), anti-phospho-ERK (1:2000), anti-ERK (1:1000) and anti-β-actin antibodies were used as primary antibodies. Proteins bound by the primary antibodies were visualized with an appropriate secondary antibody (1:5000) and then detected by an ECL Chemiluminescence kit (Vazyme, E411-05). Protein bands were quantified using NIH ImageJ software (available in the public domain) and detected using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).

Protein extraction from exosomes performed using radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Sigma-Aldrich, St. Louis, MO). The protein samples were separated by 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (Beyotime, Shanghai, China) and subsequently transferred onto PVDF membranes (Life Technology, Waltham, MA). After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with the appropriate primary antibodies, CD63 (1:1000 dilution, CST, #52090), CD9 (1:500 dilution, CST, #98327), and Alix (1:1000 dilution, CST, #92880). Following the primary antibody incubation, the membranes were incubated with the corresponding secondary antibody for an additional 1 hour at room temperature. Protein bands were visualized using an ECL chemiluminescence kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions.

The TNC tissues were homogenized in radioimmunoprecipitation (RIPA) lysis buffer (P0013B, Beyotime) using an electric homogenizer and supplemented with a mixture of phenylmethylsulfonyl fluoride (PMSF, ST506, Beyotime) and protease phosphatase inhibitors (P1045, Beyotime). The homogenate was then incubated at 4 °C for 2 h. After incubation, the homogenate was centrifuged at 16,000 rpm and 4 °C for 20 min, and the resulting supernatant was collected as the whole-cell protein extract. The protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (P0010, Beyotime). Equal amounts of protein were loaded onto a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrophoresed. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was blocked with 5% fetal bovine serum (BSA) at 37 °C for 2 h. Primary antibodies (Supplementary Material 1) were incubated overnight at 4 °C. The membrane was washed three times with tris-buffered saline Tween-20 buffer (TBST) and incubated with a horseradish peroxidase (HRP)-labeled secondary antibody (Supplementary Material 1) at 30 °C for 1 h. Immunoblotting was performed using a blot test kit (SuperFemto ECL Chemiluminescence Kit, Vazyme, E423-02), and the protein bands were visualized using an imaging system (Tanon-5200, China).