Rabbit anti acsl4

Total protein was extracted with cell lysis buffer (Solarbio, Beijing, China). Proteins were separated by 10% SDS‐PAGE and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% non‐fat milk (Sangon Biotech, Shanghai, China) for 2 h, then incubated with primary antibodies overnight at 4°C, and finally incubated with HRP‐conjugated secondary antibodies for 2 h at room temperature. Next, immunoreactive bands were detected using an enhanced chemiluminescence kit (Bio‐Rad, Hercules, CA, USA). β‐Actin was used as a loading control. The primary antibodies included mouse anti‐MAP2 (Millipore; 1:1000), mouse anti‐Tuj1 (Millipore; 1:1000), rabbit anti‐GFAP (Millipore, 1:1000), rabbit anti‐Acsl4 (Abcam; 1:1000), rabbit anti‐Akt (Cell Signaling Technology, Danvers, MA, USA; 1:1000), rabbit anti‐phospho‐Akt (Cell Signaling Technology; 1:1,000), rabbit anti‐PI3K (Abcam; 1:1000) and mouse anti‐β‐actin (Abcam; 1:1000).

The cells were washed twice with PBS, lysed in a 100 μL protein lysis buffer (RIPA: PMSF = 100:1, Solarbio, Beijing, China), and centrifuged at 12,000 rpm at 4 °C for 15 min, and then the supernatant was frozen at −80 °C. The protein concentration was determined using BCA (Beyotime, Shanghai, China), in which equal amounts of protein (60 μg) were subjected to SDS-PAGE on a 10% or 12% gel. The separated proteins were electrophoretically transferred to PVDF membranes and 5% degreased milk was used asblocking agent for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase-labeled antibodies for 1 h at room temperature. The signals were detected using an enhanced chemiluminescence reagent (Bio-Rad 170-5060, Hercules, CA, USA). The primary antibodies included mouse anti-GADPH (1:1000, ABCAm, Boston, MA, USA), rabbit anti-FTH1 (1:2000, ABCAm, Boston, MA, USA), rabbit anti-HO-1 (1:2000, CST, Danvers, MA, USA), rabbit anti-ACSL4 (1:10,000, ABCAm, Boston, MA, USA), and rabbit anti-GPX4 (1:2000, CST, Danvers, MA, USA).

Astrocytes isolated from E15 and cultured on poly-D-lysine-coated coverslips were stained with 5 μM MitoTracker DeepRed to label mitochondria. Alternatively, cells were transfected with ER-dsRed expression vector 1 day prior to fixation to label ER. Cells were fixed with 4% paraformaldehyde for 15 min and were then permeabilized by ice-cold methanol for 5 min, followed by PBS rinse for 10 min. The cells were then blocked with 5% bovine serum albumin in PBS with 0.3% TritonX-100 for 1 h, followed by primary antibody [rabbit-anti-HSP60 (CST), rabbit-anti-LC3B (CST), rabbit-anti-LAMP1 (CST), rabbit-anti-ACSL4 (Abcam), mouse-anti-STAT3 (CST), both diluted at 1:200 in the blocking buffer] incubation at 4°C overnight. Then, the cells were incubated in appropriate secondary antibodies [donkey-anti-rabbit-AF488, donkey-anti-rabbit-647, donkey-anti-mouse-488, donkey-anti-mouse-AF568 or donkey-anti-mouse-AF647 (Thermo), all diluted at 1:200 in the blocking buffer] for 1 h at room temperature. After washing three times in TBST, the cells on the coverslip was mounted using Prolong Gold with DAPI (Thermo) and subjected to confocal microscopy observation using the Zeiss LSM880 with Airyscan system. Images were captured using a 63x/1.4NA oil immersion objective. The colocalization analysis was performed with Fiji software using the Coloc 2 plugin.