Figure 1.

The production and export of bilirubin in human cells. (a) Time course of bilirubin released from cells in culture medium. HEK293T, HepG2, and HeLa cells were incubated with FCS-free VP-SFM medium plus 2.0 mg/ml bovine serum albumin (BSA). At the indicated time, aliquots of the media were withdrawn and incubated with recombinant his-tagged UnaG (2 μg/ml) for 1 h, followed by shaking with nickel ion beads for 30 min. After washing the beads twice with 10 mM Tris-HCl, pH 7.5, containing 150 mM NaCl (TBS), UnaG was eluted with TBS containing 300 mM imidazole. The fluorescence in elutes was examined by fluorospectrophotometry. (b) The production of bilirubin in K562 cells. K562 cells were incubated without or with 20 μM haemin, under conditions similar to those above. Bilirubin in the medium was estimated using fluorophotometry. (c) Immunoblot analysis of proteins involved in the generation of bilirubin. The cellular proteins of the indicated human cells were analysed by SDS-PAGE and electroblotted onto a PVDF membrane. The immunoblotting was performed with primary antibodies for HO-1, HO-2, BLVRA, BLVRB, and actin.