4.6. Western Blot Analysis

The liver tissues (100 mg) were homogenized in 500 µL of RIPA buffer [25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, Complete Mini protease inhibitor cocktail (Roche Diagnostics, Meylan, France)] with Dounce homogenizer. The homogenates were clarified by centrifugation at 12,000 rpm for 10 min at 4 °C. The protein contents of the supernatants were measured with DC Protein Assay Kit (Bio-Rad Inc.). The same amount of protein from each sample was loaded onto 12% or 14% SDS-PAGE and transferred to nitrocellulose membranes (BioTrace NT, Pall Life Sciences, Port Washington, NY) and probed with rabbit polyclonal antibodies produced against mouse ferrochelatase (Fc 1:1000; Novus Biologicals), mitochondrial ferritin (FTMT 1:1000; Thermo-Fischer Scientific Inc.), ferritin heavy chain (FTH 1:1000; Cell Signaling Technology Europe, Leiden, The Netherlands), NFS1 (1:1000; Novus Biologicals), transferrin receptor-1 (TfR1 1:1000; Thermo-Fischer Scientific Inc.) and alpha-1 antitrypsin (A1AT 1:1000; Abcam, Cambridge, UK). β-actin (1:2000; Sigma-Aldrich Kft.) was used as loading control. Goat anti-rabbit (H + L) HRP-conjugate was used as secondary antibody (1:3000; Bio-Rad Inc.). Protein detection was carried out with WesternBright ECL chemiluminescent substrate (Advansta Inc., San Jose, CA). All experiments were repeated at least three times. Optical densities of the Western blots were calculated by ImageJ software (https://imagej.nih.gov/ij) and expressed as percentage of target gene/β-actin abundance.