2.6. Western Blot Analysis

Cells were grown in YPD medium at 30 °C overnight and diluted to an OD₆₀₀ of 0.1, in YPD medium containing 150 μM BPS to deplete iron. Cultured cells were harvested, diluted to an OD₆₀₀ of 0.1 in YPD medium containing 150 μM BPS with or without 100 μM FeCl₃, and incubated at 30 °C for 12 h. Cells were harvested by centrifugation and resuspended in a protein extraction buffer comprised of 50 mM HEPES KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycolate, 1 mM PMSF, and a protease inhibitor cocktail (Sigma, Munich, BY, Germany). Cell lysates were prepared by bead-beating, and the protein concentration was determined by the Bradford assay [17]. Total proteins were separated on a sodium dodecyl sulfate–polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma, Munich, BY, Germany). Protein detection was performed using an anti-GFP rabbit polyclonal antibody (Proteintech, Rosemont, IL, USA) or an anti-FLAG (Abcam, Cambridge, UK) rabbit polyclonal antibody. A mouse anti-rabbit IgG horseradish peroxidase conjugate (Santa Cruz, Dallas, TX, USA) was used as a secondary antibody, followed by visualization by chemiluminescence.