Figure 7.

Effect of siRNA knockdown of ferritin H- and L-chain on transferrin receptor and iron uptake in LECs. (A) TfR Western blot. Twenty-four hours after transfection with a nontargeting control, FHsiRNA, or FLsiRNA, the cells were rinsed and incubated in serum-free MEM for 24 hours. After lysis in RIPA buffer and centrifugation, supernatants were concentrated on centrifugal filtering columns. Twenty-five micrograms of protein from each sample was loaded onto an 8% denaturing Tris-tricine gel, along with 6 μg of purified human placenta TfR as the positive control. Transferrin receptor was detected after Western transfer using a monoclonal mouse anti-human TfR, a secondary HRP antibody and ECL. Blots were reprobed with a 1:500 dilution of HRP-goat anti-human β-actin which was used as a loading control. The blot shown is representative of three experiments and data expressed as the mean ± SEM. *Significantly different from control (P < 0.01); ANOVA and Tukey's test. (B) Iron uptake. Transfected cells were incubated with 240 μg/mL ⁵⁹Fe-Tf for 20 hours in serum-free DMEM, lysates prepared, and radioactivity counted in the cytosol fraction using a gamma counter. Radioactivity in each sample was normalized to protein and data are expressed as a percentage of control cells transfected with nontargeting siRNA. Data are shown as the mean ± SEM of eight samples. *Significantly different from control (P < 0.05); ANOVA and Tukey's test.