More ferritin L is secreted when its synthesis is elevated. (A) Untransfected HepG2 cells were left untreated (Ctrl) or treated with 10 μM FeSO4/10 μM 8-hydroxyquinoline (FeSO4) during the first 30 minutes of the methionine starvation period. H3 and L1 cells are stable, transfected HepG2 cells expressing high levels of ferritin H or L, respectively. Cells were radiolabeled for 30 minutes and chased in complete medium overnight. The cell lysate (C) and media (M) were immunoprecipitated using polyclonal antibodies to ferritins and resolved by SDS-PAGE and fluorography. Treatment with FeSO4 increased synthesis of both ferritin H and L chains, but little secretion occurs. The stable cell line H3 synthesizes increased ferritin H, but only a trace amount is secreted. The stable cell line L1 synthesizes an increased amount of ferritin L, and some is secreted. A high-molecular-weight protein coimmunoprecipitates with ferritin L (arrow). The position of molecular mass markers is shown on the left. (B) A darker exposure of a separate labeling experiment (as in A) shows that untreated HepG2 cells (Ctrl) secrete some ferritin L. (C) HepG2 cells were left untreated (Ctrl) or treated with IL-1β at either 5 or 10 ng/mL at the outset of methionine starvation (60 minutes prior to labeling) and continuing throughout the labeling (30 minutes) and chase (overnight) periods. Note that this exposure is darker than in panel A, as some ferritin L is visible in the media of Ctrl cells.
