Horse radish peroxidase conjugated antibody for β actin

K562, a human erythroleukaemia cell line, was cultured in Iscove’s modified DMEM (Dulbecco’s Modified Eagle’s medium; IMDM), supplemented with 10 %heat-inactivated FBS, 100 μg/ml streptomycin and 100 units/ml penicillin, [30 (link)]. Cells were kept in serum-free medium overnight prior to treatment with EPO (3 units/ml). EPO was a kind gift from Dr Vinod Pullarkat (City of Hope, Duarte, California). LY294002, PD98059, SB203580, SP600125 and R59949 were purchased from EMD Millipore and used at concentrations as previously described [31 (link)]. Drugs were used at the indicated final concentrations and were obtained from vendors as noted: fenofibrate (100 μM) and GW6471 (5 μM) were purchased from Sigma–Aldrich; Ro31 (33 μM) and Ro32 (31 μM) were purchased from Tocris. Cells were pre-incubated with drugs for 30 min prior to treatment with EPO. Primary antibodies for HIF-1α (1:250) and PPARα (1:250) were purchased from Santa Cruz Biotechnology and Abcam respectively. Horseradish peroxidase (HRP)-conjugated antibody for β-actin and secondary antibodies for HIF-1α and PPARα were purchased from Sigma–Aldrich. The miR-214 mimics, anti-miR-214 inhibitors and appropriate controls were purchased from Shanghai GenePharma as well as from Exiqon, designated as miR-214 inhibitor and locked nucleic acid (LNA)-miR214 inhibitor respectively.