Anti ftl 10727 1 ap

Erastin (HY-15763), Ferrostatin-1 (HY-100579), Deferoxamine (HY-B0988), and Necrosulfonamide (HY-100573) were purchased from MedChemExpress (MCE; Shanghai, China). Sorafenib (S7397), ZVAD-FMK (S7023), Bafilomycin A1 (S1413), and RSL3 (S8155) were obtained from Selleckchem (Houston, TX, USA). N-acetyl-L-cysteine (NAC, S0077) was from Beyotime (Shanghai, China). Brusatol (Bru, MB7292) was obtained from Meilunbio (Dalian, China). Tertiary butylhydroquinone (tBHQ, 112941) was obtained from Sigma (Shanghai, China). Antibodies raised against GPX4 (ab125066), NRF2 (ab62352), 4-HNE (ab46545), NQO1 (ab34173), and β-actin (ab6276) were obtained from Abcam (Cambridge, MA, USA), anti-SLC7A11 (NB300-318) was from Novusbio (Centennial, CO, USA), anti-FTL (10727-1-AP) was from Proteintech (Shanghai, China), and anti-GSTZ1 (GTX106109) was from GeneTex (San Antonio, CA, USA).

Cells were washed twice with PBS and subsequently lysed with RIPA buffer containing protease and phosphatase inhibitors (P0013B and P1045, Beyotime, China) on ice for 30 min. Proteins in the lysates were separated by SDS-PAGE and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies, including anti-TFRC (66180-1-Ig, Proteintech, China), anti-FTH1 (49644, Signalway Antibody, USA), anti-FTL (10727-1-AP, Proteintech, China), anti-POLQ (Fab4305, Fenghui Antibody, China), anti-RAD51 (67024-1-lg, Proteintech, China), anti-phospho-Histone H2AX(Ser139) (AF5836, Beyotime, China), anti-α-tubulin (66031-1-Ig, Proteintech, China), and anti-β-actin (66009-1-Ig, Proteintech, China), at 4 °C overnight. Unbound antibodies were removed by washing the membranes three times with TBST. Goat anti-rabbit (LF102, Epizyme, China) or anti-mouse (7076 S, Cell Signaling Technology, USA) secondary antibodies were applied at a 1:5000 dilution and incubated at room temperature for 1 h. After another three washes with TBST to remove unbound antibodies, the HRP signaling was detected using an ECL substrate (P0018AM, Beyotime, China) in an illuminance imaging system (Tanon5200, Tanon, China).