Inhibition of ferritin L secretion by serum is rapid and cotranslational. (A) Serum acts during the starvation or labeling periods but not the chase period. AdX-FL–infected HepG2 cells were treated in 4 different conditions as follows: starved, labeled, and chased in medium lacking serum (lanes 1-2); starved and labeled in medium lacking serum but chased in medium with 30% fetal bovine serum (lanes 3-4); starved and labeled in the presence of 30% fetal bovine serum but chased in medium lacking serum (lanes 5-6); or starved, labeled, and chased in medium with 30% fetal bovine serum (lanes 7-8). The presence of serum during labeling—and not during the chase period—results in inhibition of ferritin L secretion. This result also indicates that serum in the chase medium does not affect the immunoprecipitation of ferritin L. (B) Serum inhibit the secretion of ferritin L rapidly and cotranslationally. Some cells were incubated in media lacking serum throughout the experiment (lanes 1-2). Fetal bovine serum was added at a 30% concentration to AdX-FL–infected HepG2 cells either 60 minutes prior to labeling with ³⁵S-methionine (at the outset of the methionine starvation period) (lanes 3-4) or 2 minutes prior to labeling (lanes 5-6) and continued through the 5-minute labeling period in both cases. At the onset of the chase period, all cells were washed in phosphate-buffered saline and chased in serum-free complete medium for 2 hours. The cell lysates (labeled C) and media (M) were immunoprecipitated with polyclonal antibodies against ferritins.