Anti hepcidin

Cells were collected, washed 3 times with ice-cold PBS, and lysed on ice with lysis buffer (10 mM Tris pH 7.5, 130 mM NaCl, 1% NP-40, 10 mM NaPPi, 1 mM PMSF, 0.1 mM Na₃VaO₄). Lysates were transferred to microcentrifuge tubes and precleared by centrifugation at 11,900×g for 15 min at 4°C. Protein concentrations were determined using Bradford reagent according to the manufacturer’s instructions. β-actin was used as an internal reference control. An equal amount of total protein extracted from cultured cells was separated by 12% SDS-PAGE and transferred to PVDF membranes (EMD Millipore Corporation, Billerica, MA, USA). Primary antibodies and HRP-conjugated appropriate secondary antibodies were used to detect the designated proteins. The bound secondary antibodies on the PVDF membrane were reacted with the ECL detection reagents (Beyontime, Institute of Biotechnology, Jiangsu, People’s Republic of China) and exposed to X-ray films (Kodak, Tokyo, Japan). The result was analyzed using ImageJ 1.46r software (National Institutes of Health, Bethesda, MD, USA). Anti-hepcidin, anti-BMP6, anti-SMAD4, anti-TfR2 (Abcam, Eugene, OR, USA), anti-NF-κB (Protein Tech, Chicago, IL, USA), and β-actin monoclonal antibody (LiankeBio, Hangzhou, People’s Republic of China) were used in the experiments.