Anti gss

Collected cells were homogenized in lysis buffer (5% SDS, 10 mM EDTA, 50 mM NaCl, 10 mM Tris–HCl). Protein concentrations were determined using pierce BCA protein assay (Thermo Fisher, Rockford, IL, USA). Proteins were resolved using SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane. Membranes were blocked in 5% milk, incubated with primary antibody at a concentration of 1:1000, then incubated with secondary antibody at a concentration of 1:10,000 and read using ECL regent (Bio-Rad, Richmond, CA, USA). Antibodies anti-GCLM, anti-GSS, anti-GCLC, anti-GSR, anti-GPX1, anti-SOD1, anti-SOD2, anti-TKT, anti-G6PD, anti-CGL and anti-Actin were purchased from Abcam. Anti-NRF2 and anti-CBS antibody was purchased from Cell Signaling Technology.

The smashed fresh brain tissues were selected blindly and RIPA Lysis Buffer (Beyotime, Haimen, China) and protease phosphatase inhibitors (PMSF, Beyotime) were mixed to fully ground. The protein concentration was measured using a bicinchoninic acid assay (Beyotime). The belt was transferred to the polyvinylidene fluoride membrane (Beyotime) after an electrophoresis process. Membranes were blocked with 5% skim milk blocking buffer at 37°C for 2 h and incubated with the following ferroptosis-rand BBB-related primary antibodies: anti-SLC7A11 (Abcam, Cambridge, UK; 1: 5,000), anti-GPX4 (Abcam; 1: 1,000), anti-PDGFR-β (Solarbio; 1: 1,000), anti-TFR1 (Abcam; 1: 5,000), anti-GSS (Abcam; 1: 5,000), anti-PI3K (Abcam; 1: 1,000), anti-p-PI3K (Abcam; 1: 1,000), anti-Akt (Abcam; 1: 10,000), anti-p-Akt (Abcam; 1: 1,000), and anti-β-actin (Abcam; 1: 5,000) as an internal control, at 4°C overnight in a thermostat shaker. After being washed by TBST (Tris-HCI buffer salt solution+Tween) buffer, all membranes were incubated with the second antibodies (Proteintech, Rosemont, IL, USA; 1: 5,000) at 37°C for 2 h. Immunoreactive membranes were processed with a chemiluminescence assay (Beyotime) (Wang et al., 2020 (link)). ImageJ software was used for analysis.