Adenovirus-transduced HepG2 cells synthesize and secrete increased amounts of ferritin L. (A) HepG2 cells were uninfected (Ctrl) or infected with AdX-GFP (GFP), AdX-FH (FH), or AdX-FL (FL). After 72 hours, the cells were labeled with ³⁵S-methionine for 15 minutes and chased in complete medium for one hour. The cell lysate (C) and media (M) were immunoprecipitated using polyclonal antibodies to ferritins. Adenovirus infection per se does not affect synthesis or secretion of ferritins (compare GFP with Ctrl). Increased synthesis and secretion of ferritin L occurs from AdX-FL–infected cells. A high-molecular-weight protein coimmunoprecipitates with ferritin L (arrow). There are 2 proteins of slightly higher molecular weight that coimmunoprecipitate with ferritin H (>) and L (^*) in cell lysates, and 2 other proteins coimmunoprecipitate with ferritin L in the medium (#). (B-D) Identification of proteins that coimmunoprecipitate with ferritin H and L in AdX-FH– and AdX-FL–infected HepG2 cells. (B) In the top panel, HepG2 cells infected with AdX-FH were lysed and immunoprecipitated using polyclonal antibodies against ferritins before Western blotting using polyclonal antibodies. In the bottom panel, HepG2 cells infected with AdX-FL were lysed and immunoprecipitated using monoclonal antibody against ferritin L before Western blotting using polyclonal antibodies. The coimmunoprecipitated proteins (denoted by > and ^*) are detected by immunoblotting. (C) Western blot showing that the high-molecular-mass protein (arrow) in AdX-FL–infected HepG2 cells (labeled C) comigrates with commercially available purified human liver ferritin (P). The cell lysate of HepG2 infected with AdX-FL was immunoprecipitated with polyclonal antibodies against ferritin, electrophoresed alongside 2 μg purified ferritin, and subjected to Western blotting using polyclonal antibody against ferritins. (D) The high-molecular-mass protein migrates as ferritin L monomers after treatment with ferrous sulfate. HepG2 cells infected with AdX-FL were labeled with ³⁵S-methionine, and the cell lysate was immunoprecipitated with polyclonal antibodies against ferritins. The beads containing the washed immunoprecipitates of the cell lysates were separated into 2 aliquots. One aliquot was left untreated (-) while the other aliquot was rotated in 0.5 mL of 100-μM FeSO₄ for 30 minutes at room temperature (+). After treatment with ferrous sulfate, the amount of the high-molecular-weight protein (arrow) is decreased and the amount of the ferritin L monomer is increased.