Be0102

Tissue or cell samples were homogenized in ice-cold lysis buffer (Beyotime) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland) and then centrifuged at 4 °C, 12,000×g to remove fat layers and debris. Protein concentration was determined by a BCA protein assay kit (Beyotime). The proteins were denatured by boiling at 95 °C in Laemmi loading buffer (Yeasen, Jiangsu, China) for 5 min. The proteins were separated by SDS-PAGE, transferred onto cellulose acetate membranes or PVDF membranes, and immunoblotted for target proteins. Blots were developed with super sensitive ECL substrate (Beyotime) using a chemiluminescent imager (Tanon 5200, Shanghai, China). The antibodies are listed as follows: anti-FTH (1:1000, Abcam, Cambridge, UK, ab65080), anti-HIF1α (1:500, Santa Cruz Biotech, Dallas, TX, USA, sc-13515), anti-β-actin (1:10,000, Abbkine, Wuhan, China, ABL01010), anti-β-tubulin (1:10,000, Abbkine, ABL01030), anti-mouse secondary antibody (1:10,000, Easybio, Beijing, China, BE0102), anti-rabbit secondary antibody (1:10,000, Easybio, BE0101).

Tissues were homogenized in lysis buffer (P0013B, Beyotime, Haimen, China) containing protease inhibitor cocktail (P0013B, Beyotime, Haimen, China). The homogenates were centrifuged (4 °C, 15871 rcf, 20 min), and the supernatant was collected. Approximately 20 μg of proteins was loaded on a Tris-glycine gel. Proteins were transferred onto PVDF membranes, blocked, and stained overnight using the following primary antibodies: anti-CHD8 (1:1000, ab114126, Abcam), HRP-conjugated anti-GAPDH (1:5000, BE0034, Easybio, Beijing, China), anti-SLC6A19 (1:5000, ab180516, Abcam), and anti-SLC7A8 (1:5000, ab75610, Abcam). Membranes were washed and stained with HRP-conjugated secondary antibodies (1:5000, BE0101 and BE0102, Easybio, Beijing, China) before washing and development with enhanced chemiluminescence (ECL) substrate for Western blotting (BE6706, Easybio, Beijing, China).