Ab222320

The proteins were separated by 10% SDS–PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked in 5% BSA for 2 h at room temperature. The membranes were washed once for 5 min with PBST and incubated with primary antibodies against Akt (Abcam, ab179463), p‐AKT (Abcam, ab192623), STAT3 (Abcam, ab68153), p‐STAT (Abcam, ab76315), GAPDH (Proteintech, 60004‐1‐Ig), CDK7 (Abcam, ab256787), β‐Tubulin (Proteintech, 10068‐1‐AP), MLKL (Abclonal, A13451), p‐MLKL (Abclonal, AP0949; Boster, P00535), RIPK1 (Abclonal, A7414), p‐RIPK1 (Abclonal, AP1115), RIPK3 (Abclonal, A5431), and p‐RIPK3 (Abcam, ab209384; Abcam, ab222320) overnight at 4°C. The membranes were washed three times for 15 min with PBST and incubated with HRP‐conjugated secondary antibodies for 90 min at room temperature. The membranes were washed three times for 15 min each with PBST and visualized using ECL (Biosharp).

Abcam provided rabbit anti-MLKL (ab184718), rabbit anti-phospho-RIP3 (T231, S232) (ab222320), rabbit anti-phospho-MLKL (S345)(ab196436), rabbit anti-TNFR1 (ab68160), rabbit anti-TRADD (ab110644), and rabbit anti-CIAP2 (ab32059) antibodies. Rabbit anti-RIP1 (3493 s) and rabbit anti-IκBα (9242) antibodies were purchased from Cell Signaling Technology. The mouse anti-GAPDH (AC033) antibody was purchased from AbClonal. Sigma provided the rabbit anti-RIP3 (PRS2283) antibody. Santa Cruz Biotechnology offered mouse anti-β-actin (sc-47778) antibody. Calbiochem (San Diego, CA, USA) offered Benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD; 627610). Sigma-Aldrich offered propidium iodide (PI; P4170). Murine TNF-α (PMC3015) was purchased from Thermo Fisher. Ketamine (1707031) was obtained from Gutian Pharmaceutical Co. Ltd.

For histology, specimens of fresh small intestine were fixed in 4% paraformaldehyde. After dehydrated in ethanol, the intestinal tissue was embedded with paraffin and was stained with hematoxylin and eosin (H&E). To evaluate intestinal injury, Chui’s score system was used as previously described^{33 (link)}. For immunohistochemistry (IHC), paraffin sections were deparaffinized with xylene and rehydrated with the concentration gradient of ethanol. Then, antigen retrieval was conducted and the samples were incubated with primary antibodies for p-MLKL (phospho S345, ab196436, Abcam) or p-RIPK3 (phospho T231 + S232, ab222320, Abcam) and secondary antibodies (Abcam) and developed with DAB Substrate kit. After visualized with the DAB substrate kit, the sections were rinsed, dehydrated, cleared, and mounted. Finally, the IHC was quantified with Image J software (US National Institutes of Health).
For immunofluorescence staining, frozen sections were cut and mounted on slides. After blocked with 2% bovine serum albumin in PBS at 37 °C for 1 h, the sections were incubated with 1:100 dilutions of primary antibodies, including ZO-1 (61-7300, Invitrogen) and occludin (ab216327, Abcam) and the nuclei were counterstained with DAPI according to the manufacturer’s instructions.