Enhanced ecl kit

RIPA lysis buffer (RIPA: PMSF = 100:1) was used to extracted the inner protein of LUAD cells. After measured all densities, the prepared proteins (30 µg/lane) were orderly separated by SDS-PAGE (10% or 12%) and then transferred onto pre-cut PVDF membranes. After blocking in skimmed experimental milk (5%) for 2 h at 37 ℃, the targeted membranes were incubated with preconfigured primary anti-FTH (#4393; 1:600; CST; USA), PTGS2 (#12,282; 1:600; CST; USA), DMTI (#15,083; 1:800; CST; USA), SLC7A11 (ab216876; 1:800; Abcam; USA), GPX4 (ab125066; 1:800; Abcam; USA) and β-actin (#4970; 1:3000; CST; USA) overnight at 4 °C. Next day, these were treated with corresponding secondary antibody. Finally, protein signals on the finished membrane were visualized by enhanced ECL kit (4 A Biotech, China).

The total protein of HCC cells and HCC tissues was extracted as described previously.²⁰ (link) The mitochondrial, cytoplasmic, and nuclear fractions of HCC cells were obtained using commercial kits (Keygene, Nanjing, China). Protein samples were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and western blotting. After transferring the proteins to a membrane and blocking, the blots were sequentially incubated with primary and secondary antibodies. The bands were visualized using an enhanced ECL kit (4A Biotech, Beijing, China). Images were captured using a Tanon5200 imaging system (Tanon, Shanghai, China).