NEO1 is required for furin-mediated cleavage of HJV in HepG2 cells. (A) Diagram of HJV and furin-cleaved soluble HJV (sHJV). PM, plasma membrane. (B) Knockdown of endogenous NEO1 by small interfering RNA (siRNA) abolishes HJV shedding from HepG2-HJV cells. HepG2-HJV cells were transfected with control or NEO1-specific siRNA. Cell surface proteins were biotinylated at 4°C, followed by pull-down of the biotinylated proteins using streptavidin agarose beads. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated conditioned medium (CM) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunodetection by using anti-NEO1 FNIII 1-6, furin, HJV, Na⁺K⁺ ATPase, and β-actin antibodies. Experiments were repeated 3 times with consistent results. (C) fHjv and fHjv^A183R shedding from Hep3B cells. Hep3B cells were transiently transfected with pCMV9-fHjv or pCMV9-fHjv^A183R construct. Total cell extracts and a fraction of concentrated CM were subjected to SDS-PAGE and immunodetection by using anti-NEO1 FNIII 1-6, FLAG, β-actin, and HJV antibodies. Experiments were repeated three times with consistent results. (D) A model for the induction of hepcidin expression by hepatocyte Neo1 via its interaction with Hjv.