Anti l ferritin

Liver tissue and hepaRG cells underwent lysis by sonication (Next Advance Inc., Averill Park, NY) in radio‐immunoprecipitation assay (RIPA) buffer (50 nM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100). Anti‐L‐Ferritin or anti‐α‐Tubulin (T6199; Sigma‐Aldrich, St. Louis, MO) antibody was used. Horseradish peroxidase‐conjugated secondary antibodies (Santa Cruz Biotechnology, Inc.) were used, and the protein bands were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Liver and spleen tissues or cells extracts were prepared using RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 50 mM Tris pH 8.0, 50 mM dithiothreitol, 0.01 mg/mL leupeptin, and Protease Inhibitor Cocktail (Roche) or using a lysis buffer (200 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and Complete Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy). Protein was quantified by BCA assay (ThermoScientific-Pierce, Spinea, Italy). Samples (40–50 µg) were separated on 10–14% SDS-PAGE and transferred to Hybond-P Membrane (GE, Milan, Italy). The primary antibodies used for immunoblotting were: anti-l-ferritin (SIGMA #F5012), anti-GAPDH (ORIGENE #TA802519), and anti-actin (ORIGENE #811000). After incubation with horseradish peroxidase-conjugated secondary antibodies, membranes were developed with SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific-Pierce) and visualized with Lycor Odyssey instrument. Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD, USA) normalized against actin (for HepG2, J774 and Spleen) and GAPDH (for liver samples).