Monocyte-derived hepcidin causes ferroportin degradation in vitro. (A) Western blot of THP-1 cells transfected with EmGFP/CAT expression vector (lanes 1–4; as control) or EmGFP/ferroportin expression vector (lanes 5–8). Cells were either left untreated (control, lanes 1 and 5), cotransfected with a hepcidin expression vector for another 12 hours (lanes 2 and 6), treated with natural human hepcidin (1 μg/mL) for 3 hours (lanes 3 and 7), or stimulated with LPS 1 μg/mL for 6 hours (lanes 4 and 8). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown. (B) Immunofluorescence of THP-1 cells: Cells were transfected with ferroportin/EmGFP expression vector and left untreated (lane 1) or stimulated with LPS (1 μg/mL) for 6 hours (lane 2). PKH-26, a red fluorescence cell linker, was used for cell membrane staining. Ferroportin/EmGFP fusion protein showed green fluorescence. Colocalization of both results in a yellow staining. The size of the images is 360 × 360 μm displayed in original resolution of 1024 × 1024 pixel colored in 8 bit using the ×630 magnification.