Hemojuvelin protein exists in both cell-associated and soluble forms. HEK293 and Hep3B cells were transfected with pcDNA3.1(+) or pcDNA-HJV in 6-well tissue culture plates and incubated overnight, followed by a 24-hour incubation in serum-free medium (2 mL/well). Whole-cell lysates were collected in 150 μL NETT buffer per well and 30 μL cleared total protein solution was analyzed. Conditioned cell culture media (2 mL/sample) were filter concentrated (5-kDa cut-off) and concentrates equivalent to 800 μL starting material were analyzed. Western blots after reducing SDS-PAGE were probed with anti-G3pep2-3 antibody. Arrows indicate cell-associated hemojuvelin (apparent molecular weight, 46 kDa) in both human liver protein extracts (no. 1 and no. 2) and whole-cell lysate of HEK293 cells transfected with pcDNA-HJV (pHJV), but not in HEK293 cells treated with control vector (pcDNA). Soluble hemojuvelin (apparent molecular weight, 44 kDa) is indicated by the asterisk and seen in conditioned cell culture media from HEK293 and Hep3B cells transfected with pcDNA-HJV (pHJV), as well as in conditioned media from Hep3B cells transfected with control vector (pcDNA) but not in media from HEK293 cells treated with control vector (pcDNA).