Anti p rip3

Total proteins were harvested from the cortex and hippocampus. The proteins were transferred to polyvinylidene fluoride membranes (Millipore) that were blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam), anti‐p‐MLKL (Abcam), anti‐tumour necrosis factor‐α (TNF‐α) (Abcam), anti‐IL‐1β (Abcam), anti‐IL‐6 (Abcam) and anti‐β‐actin (Cell Signaling Technology). After incubation with secondary antibodies (Solarbio), the positive bands were visualized using an ECL substrate (Solarbio). For immunohistochemical analysis, brain tissue sections were incubated overnight with the following primary antibodies at 4℃: anti‐p‐RIP3 (Abcam) and anti‐p‐MLKL (Abcam). Subsequently, a PV9000 kit (ZSGB‐BIO) was used for follow‐up. For immunofluorescence analysis, brain tissues were blocked with goat serum for 1 hour and incubated with primary antibodies against p‐RIP3 (Abcam), p‐MLKL (Abcam), NeuN (Abcam), Iba‐1 (Wako; Servicebio) and GFAP (Servicebio) overnight at 4℃. Then, the tissues were incubated with Alexa Fluor® 594 goat anti‐rabbit IgG and Alexa Fluor® 488 goat anti‐mouse IgG secondary antibodies (Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific).

The protein collecting and western blotting were performed according to the protocol. The primary antibodies were as follows: anti-p-PKM2 (Tyr¹⁰⁵) (cat: 3827 s, CST), anti-PKM2 (cat: 4053 s, CST), anti-Na/K-ATPase (cat: 3010 s, CST; cat: ab76020, Abcam), anti-AQP1 (cat: ab168387, Abcam), anti-KIM-1 (cat: ab47635, Abcam), cleaved-caspase3 (cat: 9664 s, CST), caspase 3 (cat: 9662 s, CST), anti-RIP1 (cat: 3493 s, CST), anti-p-RIP3 (cat: ab222320, Abcam), anti-RIP3 (cat: 15828 s, CST), anti-p-MLKL (cat: 37333, CST), and anti-MLKL (cat: 37705, CST), anti-GPX4 (cat: ab125066, Abcam), anti-p-DRP1 (cat: 3455, CST), anti-DRP1 (cat: ab184247, Abcam), anti-MYH9 (cat: 11128-1-AP, Proteintech), anti-VDAC (cat: 4866 s, CST; cat: ab14734, Abcam), anti-TBP (cat: ab818, Abcam), and anti-α- tubulin (cat: T9026, Sigma Aldrich).

Antibodies used in immunoblotting and immunofluorescence: anti-RIP3 (Cell Signaling Technology, 13526s, 1:1000), anti-p-RIP3 (Abcam, ab209384, 1:1000), anti-MLKL (Abcam, ab184718, 1:2000), anti-p-MLKL (Abcam, ab187091, 1:1000), anti-mouse p-MLKL (Abcam, ab196436, 1:5000), anti-RIP1 (BD Biosciences, 610458, 1:1000), anti-p-RIP1 (Cell Signaling Technology, 65746, 1:1000), anti-mouse p-RIP1 (Cell Signaling Technology, 31122, 1:1000), anti-ACTIN (Sigma-Aldrich, A3853, 1:5000), anti-p-ERK (Cell Signaling Technology, 9101s, 1:1000) and anti- IκB-α (Santa Cruz Biotechnology, sc-371, 1:5000). TNF-α and zVAD were purchased from R&D Systems. SMAC mimetic (LCL-161) was purchased from Adooq Bioscience. Dabrafenib and GSK’872 were purchased from Selleckchem. Necrostatin-1, lipopolysaccharide (LPS), and propidium iodide (PI) were purchased from Sigma-Aldrich. Cycloheximide was purchased from Calbiochem. Polyehylenmine was purchased from Polysciences. We purified GST-TRAIL.