Ab208910

Western blotting assays were carried out as previously described^{29 (link),30 (link)}. Briefly, tissue samples or cell lysates were prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Nantong, Jiangsu, China). Equal amounts of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes, which were blocked and incubated overnight with the following primary antibodies: anti-RIP3 monoclonal antibody (#95702, Cell Signaling Technologies) or anti-RIP3 polyclonal antibody (ab152130, Abcam, Cambridge, MA, USA), anti-phospho-RIP3 monoclonal antibody (ab205421, Abcam, Cambridge, MA, USA), anti-MLKL polyclonal antibody (#28640, Cell Signaling Technologies) or anti-MLKL monoclonal antibody (#14993, Cell Signaling Technologies), anti-phospho-MLKL monoclonal antibody (ab208910, Abcam, Cambridge, MA, USA) or anti-phospho-MLKL monoclonal antibody (#91689, Cell Signaling Technologies), or anti-β-actin monoclonal antibody (sc-47778, Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP). Immune-reactive signals were visualized by an enhanced chemiluminescence (ECL) kit (Millipore, USA) on a Syngene PXi6 Access imaging system (Frederick, MD). The band intensities were quantified using Image-Pro Plus 6.0.

Cytosolic, cytomembrane and overall extracts of protein were requested following the previous description.²⁸, ²⁹ Proteins at the site of trauma and on the contralateral side were harvested at different time and extracted using radioimmunoprecipitation assay (RIPA) buffer on ice (Sigma) and then centrifuged for 15 minutes 1000 g 4°C. The proteins underwent separation using 12% SDS‐PAGE gel and placed to polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes underwent incubation with 5% (w/v) non‐fat dried milk for 2 hours at ambient temperatures and then incubated using primary antibodies throughout the night at 4°C. The membranes underwent incubation in the antibodies below: GAPDH (1:2000, #5174; Cell Signaling Technology), RIP3 (1 µg/mL, ab62344; Abcam), p‐MLKL (1:500, ab208910, Abcam), CHMP4B (1 µg/mL, ab105767; Abcam) and FOXO1 (1:1000, ab39670; Abcam). Using an enhanced chemiluminescence detection system (GE Healthcare), we ascertained bound antibodies. Applying ImageJ software (National Institutes of Health), we studied the achieved bands’ optical density.

The frozen brain sections were permeabilized for 20 minutes with 0.2% Triton X‐100 (Sigma‐Aldrich, St Louis, MO; USA, X100); then, they underwent blocking processed with 5% normal goat serum (Millipore; S26‐LITER) and incubation throughout the night using major antibodies and then using minor antibodies for 2 hours at ambient temperatures. Nuclei underwent staining processed using DAPI. Major antibodies included RIP3 (5 µg/mL, ab62344; Abcam), p‐MLKL (1:100, ab208910, Abcam), CHMP4B (5 µg/mL, ab105767; Abcam) and FOXO1 (1‐5 µg/mL, ab39670; Abcam). Cell processing was like sections, except that they underwent initial fixing processed for 20 minutes with pre‐cooled paraformaldehyde (4%, w/v). Using an Olympus FluoView confocal microscope with appropriate emission filters (Olympus), we observed immunoreactivity.