Iron loading reduces soluble hemojuvelin release into cell culture medium. Each panel is representative of at least 3 independent experiments. HEK293 and Hep3B cells were transfected with hemojuvelin vector (pcDNA-HJV) in 6-well tissue culture plates and incubated overnight, followed by a 24-hour incubation in serum-free medium (2 mL/well) with (A) FAC concentrations ranging from 0 to 100 μM. Conditioned cell culture medium (2 mL/sample) was filter-concentrated (5-kDa cut-off) and analyzed on a reducing SDS-PAGE/Western blot probed with anti-G3pep2-3. In both Hep3B (top panel) and HEK293 cells (bottom panel), the amount of soluble hemojuvelin decreased progressively with increasing FAC concentrations. (B) Holotransferrin and apotransferrin were added to pcDNA-HJV-transfected HEK293 cells at various ratios to reach a constant total transferrin concentration of 30 μM. Conditioned cell culture medium (2 mL/sample) was extracted by cation exchange and filter-concentrated (5-kDa cut-off) before being analyzed on a nonreducing SDS-PAGE/Western blot probed with Ab112. Lane 1 shows conditioned cell culture medium from pcDNA3.1(+) vector-transfected HEK293 cells as a negative control. The amount of soluble hemojuvelin decreased progressively with increasing iron saturation of transferrin.