Pr30011

Cells that had been treated were collected and total proteins were extracted with RIPA buffer (G2002-100ML, Servicebio). Bicinchoninic Acid (BCA) method was applied to detect protein concentrations. Equivalent proteins were separated via 10% SDS-PAGE (PG111, Epizyme, Shanghai, China) and then transferred to PVDF membranes (IPVH00010, Millipore, Burlington, MA, USA). Next, incubation with primary antibodies overnight was performed at 4 °C after the membranes were blocked with a 5% skim milk powder solution at room temperature for 1 h. Following rinsing in Tris Buffered Saline-Tween (TBST, G0004-500ML, Servicebio), the membranes were incubated for 2 h at room temperature with HRP-labeled secondary antibodies (1:2000, PR30011, Proteintech, Wuhan, China). Primary antibodies against Vimentin (Vim, 1:2000, 10366-1-AP, Proteintech), N-cadherin (N-Ca, 1:2000, 22018-1-AP, Proteintech), E-cadherin (E-Ca, 1:2000, 20874-1-AP, Proteintech), ORM1 (1:1000, 16439-1-AP, Proteintech), phosphorylated PI3K (p-PI3K, 1:2000, ab278545, Abcam, Waltham, MA, USA), PI3K (1:1000, ab151549, Abcam), phosphorylated AKT (p-AKT, 1:1000, ab38449, Abcam), AKT (1:500, ab8805, Abcam), and GAPDH (1:5000, 10494-1-AP, Proteintech) were used. All antibodies were diluted in the proper proportions using an antibody diluent (WB050D, NCM Biotech, Suzhou, China).

We collected thymus for protein extraction using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China). The protein from the thymus was separated by electrophoresis. Transfer the protein to the PVDF membrane at 4 °C. After blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated with GPX4 primary antibody (ab40993, Abcam, Waltham, MA, USA) and GAPDH primary antibody (60004-1-Ig, Proteintech, Wuhan, China) at 4 °C overnight. After incubation with peroxidase-conjugated secondary antibodies (PR30011 and PR30012, Proteintech, Wuhan, China) for 1 h at room temperature. The signal was detected on an ImageQuant LAS 500 (GE, Chicago, IL, USA) using the ECL Kit (Biosharp, Hefei, China). GAPDH expression served as a loading control for quantification.

Treated cells were fixed with pre-cooled methanol for 10 min and permeabilized with 0.25% Triton X-100 for 10 min. The cells were then blocked with mixed buffer (1% w/v BSA, Sigma-Aldrich, USA; 22.52 mg/mL glycine in PBST) at room temperature for 1 h. Cells were incubated with rabbit anti-GFAP (1: 200, 16825-1-AP, Proteintech 16825-1-AP, Proteintech), rabbit anti-C3/C3b/C3c (1:200, 21337-1-AP, Proteintech), rabbit anti-EAAT2 (1:200, 22515-1-AP), and rabbit anti-Phospho-NF-κB p65 (Ser536) (1:200. 80979-1-RR, CST) were incubated at 4 °C for 12 h. Next, the samples were incubated with goat anti-rabbit IgG-HRP (1:200, PR30011, Proteintech) at room temperature and in the dark for 1 h. DAPI (4 μg/mL, Thermo Fisher, MA, USA, 62248) was used as a nucleus staining for 5 min. Finally, cells were observed for staining using a fluorescence microscope (E800 Nikon, Tokyo, Japan).