Western Blot Analysis of Iron-Regulatory Proteins

Samples were loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad) [7 (link)]. Protein was transferred onto a nitrocellulose membrane and probed for Fpn (Alpha Diagnostics, MTP11-S, 1:1000), DMT1 (Millipore, ABS983, 1:1000), Heph (Santa Cruz, SC-365365, 1:1000), TfR (Santa Cruz, sc-65882, 1:250), Tf (Abcam, ab82411, 1:1000), HA tag (Invitrogen, MA5-27915, 1:1000), ubiquitin (Protein Tech, 10201–2-AP, 1:1000) or cyclophilin B (Abcam, ab16045, 1:1000) as a loading control. Corresponding secondary antibody conjugated to HRP was used (1:5000, GE Amersham) and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham). Cellular lysate samples were normalized to cyclophilin B protein as a loading control, and then subsequently normalized to an untreated control sample within each experiment. Membrane protein samples were stained with Ponceau S and normalized to total protein as a loading control.