Western Blot Analysis of HAMP

Cells and tissues were homogenized and lysed with ice-cold RIPA buffer supplemented with a proteasome and phosphatase inhibitor (#P0013B, Beyotime, China) for 30 min, and total proteins were extracted. The procedure for standard western blotting was performed as described in a previous study [23 (link)]. Primary antibodies anti-GAPDH (1:1000, #2118; CST) and anti-HAMP (1:1000, BM5068; Boster) were used to determine the expression of the corresponding proteins. Membranes were incubated in the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at a 1:3000 ratio (Affinity Biosciences, Cincinnati, USA), and the protein bands were visualized by an ECL western blotting detection kit (Beyotime, Shanghai, China). GAPDH was used as the internal reference to normalize the protein loading. The images were quantified using ImageJ software.