Protein extraction reagent

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the loading control. Tissue was homogenized in protein extraction reagent (Keygen, China). The obtained homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The protein samples collected from the supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 8% concentration and transferred to 0.45-μm polyvinylidene difluoride membranes, which were subsequently blocked with TBST buffer for 2 h at 37 °C. The membranes were probed with primary rabbit anti-TSP1 (1:1000, MA5-13,398, Thermo Fisher Scientific, USA), rabbit anti-TSP2 (1:600, PA5-97,117, Thermo Fisher Scientific, USA), rabbit anti-TSP4 (1:1000, ab156258, Abcam, USA), and rabbit anti-GAPDH (1:2000, BA2913, Boster, USA) antibodies for 12 h at 4 °C. Next, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (1:10,000, BA1054, Boster, USA) for 1 h at 37 °C. The immunoreactive bands were visualized with a chemiluminescent substrate (Thermo Fisher Scientific, USA). The optical densities (ODs) of the bands on the Western blots were quantified using Fiji software (USA). The relative expression levels of specific proteins were normalized and calculated as the optical density of the specific protein band/OD of the GAPDH band.

Total proteins of whole intestinal tissue were extracted using Protein Extraction Reagent (KeyGENBioTECH, Nanjing, China) and the concentrations were determined with the BCA Assay Kit (KeyGENBioTECH, Nanjing, China). Equal amounts of protein were separated by SDS-PAGE and electroblotted onto polyvinylidenedifluoride membranes (PVDF) (Millipore, Bedford, MA, United States) followed by blocking with 5% fat-free milk. Then, the membranes were probed separately with rabbit anti-villin (Proteintech, 16488-1-AP, 1:2,000), anti-E-cadherin (Proteintech, 20874-1-AP, 1:2,000), anti-ZO-1 (Proteintech, 21773-1-AP, 1:2,000), anti-Cleaved Caspase-3 (CST, 9664, 1:1,000), anti-SETDB1 (CST, 93212, 1:1,000) and anti-H2AX (CST, 9718, 1:1,000) overnight at 4°C. After washing with TBST, the membranes were incubated with goat anti-rabbit secondary antibodies (Huabio, HA1001-100, 1:2,500) for 1 h at room temperature. Signals were detected using ChemiScope3500 Mini System, after that ECL was added onto the membranes and band intensity was quantified by Image J software.