Hepcidin

Total RNA was extracted from liver, kidney and bone marrow using the PerfectPure RNA Tissue kit (Gentra Systems, Minneapolis, MN, USA) and cDNAs were synthesized using high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Quantitative real time PCR (qPCR) assays were performed with power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA) as previously described [15 (link),17 (link)], using the ABI Prism 7300 Sequence detection System (Applied Biosystems, Foster City, CA). Primers for quantification of Hepcidin, HIF-2 α, EPO, EPOR and β-actin (Sigma-Aldrich, Rechovot, Israel) are summarized in (Table 1). Each sample was analyzed in triplicate in individual assays. The specificity of the reaction is derived by the detection of the melting temperatures (Tms) of the amplification products immediately after the last reaction cycle. The target genes expression value was calculated by the ΔΔct method after normalization with a housekeeping gene (β-actin). Bone marrow aspirates cells were first suspended in RNA Save solution (Biological Industries, Beit Haemek, Israel). The remaining analysis was similar to the above mentioned methods.

For quantification of specific protein expression of iron handling targets the automated, gel-free Western blot system, Simple Western™ was used (Protein Simple, San Jose, CA, USA). The antibodies used were actin (catalog #3700S) and HO-1 (catalog #70081S) from Cell Signaling Technologies (Danvers, MA, USA); transferrin (catalog #NB500-418), ferroportin (catalog #NBP1-21502), and transferrin receptor (catalog #NBP2-32945) from Novus Biologicals (Centennial, CO, USA); DMT (catalog #PA5-35136) from Thermo Fisher Scientific (Waltham, MA, USA); and hepcidin (catalog #SAB1405228) from Sigma-Aldrich (St. Louis, MO, USA).