Figure 5.

Ferritin chain degradation after siRNA knockdown. Twenty-four hours after transfection with a control nontargeting siRNA, FHsiRNA, or FLsiRNA, LECs were incubated for 18 hours with 60 μCi translabel ³⁵S-methionine. After the labeling period, the cells were either harvested (time 0) or rinsed and incubated an additional 12 or 28 hours in DMEM with 10% FBS and a large excess of cold methionine and cysteine, to prevent recycling of the radioactive label. Ferritin was immunoprecipitated from lysates with goat anti-horse ferritin and H- and L-ferritin chains separated by 12% SDS-PAGE. Radioactivity in the ferritin bands was measured and normalized to radioactivity incorporated into total cell protein. Degradation of each chain at 12 and 28 hours is expressed as a percentage of the corresponding chain and transfection treatment at time 0 (%CTL time 0). Each bar represents the mean ± SEM of four samples. *Significantly different from its time 0 counterpart (P < 0.05); ANOVA and Tukey's test.