Ripa lysis buffer

Nine mice were used to extract RNA, and nine RNA samples were collected for each brain region and the whole brain. Brain tissues (50–100 mg) were washed three times with ice-cold PBS to remove residual blood, followed by the addition of 1 mL TRIzol (Invitrogen, Life Technologies, Carlsbad, CA, USA). After homogenizing with an electric homogenizer, RNA was extracted according to the manufacturer’s instructions. Whole brain RNA was obtained by mixing the RNA samples of the seven brain regions.
A total of 15 mice were used for protein isolation. First, the same brain regions from five mice were mixed randomly to extract proteins, and then, three protein samples were obtained for each brain region and the whole brain. Next, the brain tissues were cleaned with ice-cold PBS, followed by the addition of RIPA lysis buffer (KeyGEN BioTECH, Co., Ltd., Nanjing, China) containing phenylmethylsulfonyl fluoride (1:100). The mixture was homogenized, incubated for 30 minutes at 4°C, and then centrifuged at 13,800 × g for 30 minutes at 4°C. The supernatant was harvested, and protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Whole brain protein was obtained by mixing the protein samples of the seven brain regions.

Whole-cell protein samples were prepared by homogenization in RIPA Lysis Buffer (KeyGEN BioTECH) supplemented with a protease and phosphatase inhibitor cocktail. Cell lysates containing 20 μg of protein were electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies against phospho-Akt (Cell Signaling Technology), Akt (Cell Signaling Technology), phospho-p38 mitogen-activated protein kinases (MAPK) (Cell Signaling Technology), p38 MAPK (Cell Signaling Technology), Cleaved Caspase-9 (Cell Signaling Technology), and GAPDH (CWBIO, Beijing, People’s Republic of China). After the hybridization of secondary antibodies for 1 hour at room temperature, detection was achieved by acquiring the image directly in a chemiluminescence-compatible digital imaging system (C-DiGit Blot Scanner, LI-COR Biosciences, Cambridge, UK).

Cell lysates were lysed with RIPA Lysis Buffer (KeyGEN, China) and cocktail (MCE, USA). After boiling, the lysate separation was performed with 10% SDS-PAGE (EpiZyme, China). Then transferred PVDF membranes (Millipore, USA) were blocked with 5% milk and incubated with indicated antibodies at 4°C overnight. The antibodies used in this study are listed: MTDH (Proteintech, 13 860-1-AP, 1:500), β-actin (CST, 8457 s, 1:1000), GPX4 (CST, 52455, 1:1000), and HRP Goat Anti-rabbit IgG (Abbkine, A21020, 1:10 000). The membranes were detected using ECL detection reagents (KeyGEN).