Figure 5. Pulse-chase study of hepcidin processing in primary human hepatocytes.

Cells were treated for 18 hr with 10 ng/ml BMP-9 prior to, and during the depletion and radiolabeling steps to increase hepcidin synthesis. Cells were labeled with ³⁵S-met-cys and ¹⁴C-amino acids for 1 hr then subjected to cold chase for the times indicated above the lanes, in minutes. Cell lysates and conditioned media in lane EQ are from hepatocytes labeled for 4 hr prior to processing. Cell lysates (top panel) or the corresponding media (lower panel) were immunoprecipitated with anti-mature hepcidin antibody (panel A) or anti-pro-peptide (panel B) and analyzed on SDS-tricine PAGE. Autoradiograms of the gels are shown with the molecular weight standards indicated. In the right half of the autoradiogram, cells were treated with furin inhibitor (decanoyl-RVKR-CMK) during the amino acid depletion step (1 hr) prior to and during the radiolabeling procedure.