Western Blot Analysis of Iron Regulators

Livers were washed with ice-cold PBS and dissected into pieces. Aliquots were snap frozen at liquid nitrogen and stored at −80°C. Protein lysates were obtained as described (Katsarou et al., 2021 (link)). Lysates containing 40 μg of proteins were analyzed by SDS-PAGE on 9–13% gels and proteins were transferred onto nitrocellulose membranes (BioRad). The blots were blocked in non-fat milk diluted in tris-buffered saline (TBS) containing 0.1% (v/v) Tween-20 (TBS-T), and probed overnight with antibodies against ferroportin (Ross et al., 2017 (link); 1:1000 diluted monoclonal rat anti-mouse 1C7, kindly provided by Amgen Inc), β-actin (1:2000 diluted; Sigma), Tfr2 (1:1000 diluted rabbit polyclonal; Alpha Diagnostics), or Tfr1 (1:1000 diluted mouse monoclonal, Invitrogen). Following a 3 × wash with TBS-T, the membranes were incubated with peroxidase-coupled secondary antibodies for 1 hr. Immunoreactive bands were detected by enhanced chemiluminescence with the Western Lightning ECL Kit (Perkin Elmer).