Anti ncoa4

Cell pellets were extracted using SDS extraction buffer (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% DOC, 1% NP-40, and 0.1% SDS) or 1% Triton X-100 lysis buffer (containing 10% glycerol, 150 mM NaCl, 25 mM Hepes pH 7.4, 1 mM EDTA, 1.5 mM MgCl₂, proteinase inhibitor cocktail) and gels were blotted onto nitrocellulose membranes. Antibodies used for immunoblotting were as follows: anti-LAMP (Lysosome-associated membrane glycoprotein)-1 (1:1000; Santa Cruz), anti-LAMP-2 (1:1000; Santa Cruz), anti-GBA (Lysosomal acid glucosylceramidase; 1:1000; Abcam), anti-GAA (Lysosomal alpha-glucosidase; Invitrogen; 1:1000), anti-TOMM20 (translocase of the outer mitochondrial membrane 20; 1:1000; Santa Cruz), anti-LC3A/B (microtubule-associated protein 1 light chain 3; 1:500; Cell Signaling), anti-Beclin 1 (1:1000, Cell Signaling), anti-Sequestosome1 (p62; 1:1000; Cell Signaling), anti-GPX4 (Glutathione peroxidase 4; 1:1300; Abcam), anti-ferritin heavy chain (FTH, 1:600; Cell Signaling), anti-NCOA4 (1:500; Bethyl Laboratories), and anti-β-actin (1:1,000,000; Sigma).

Renal cortex or BUMPT cells were homogenized, and the homogenates were centrifuged for the collection of the supernatants. The protein concentration was determined by BCA assay (Beyotime, catalog P0012S). The samples with equal amounts of protein were then subjected to SDS-PAGE analysis and the fractionated proteins were transferred to PVDF membranes (0.22 μm). The membranes were immersed in 5% milk for 1 h to block the background. They were incubated with diluted primary antibodies overnight at 4 °C. The primary antibody used included: anti-NCOA4 (Bethyl Laboratories, catalog # A302-272A; 1:1000), anti-FTH1 (Cell Signaling Technology, catalog # 3998S; 1:1000), anti-GPX4 (Affinity Biosciences, catalog DF6701; 1:1000), anti–NOX4 (Abcam, catalog # ab133303; 1:2000), anti–CHIP (Abcam, catalog # ab134064; 1:2000), anti-β-actin (proteintech, catalog # 66009-1-Ig; 1:5000) and anti-GAPDH (proteintech, catalog # 60004-1-Ig, 1:1000). The membranes were washed with TBST and incubated with secondary diluted antibodies (1:1000) for 1–2 h. Finally, the membranes were washed and subjected to evaluation by ECL chemiluminescence detector.