Down-regulation of TfR1 reduces the increase in TfR2 protein. (A) The 42/6 anti-TfR1 antibody interacts with TfR1 but not with TfR2. HepG2 cell lysates were immunoprecipitated with (+) or without (-) 42/6 anti-TfR1, 3B82A1 anti-TfR1, or 9F81C11 anti-TfR2 monoclonal antibodies and were analyzed by Western blot with sheep anti-TfR1/Tf serum and rabbit anti-TfR2 serum. Bands corresponding to TfR2, TfR1, Tf, and the immunoglobulin heavy chains (Ig HC) are indicated. (B-C) Treatment with 42/6 diminishes the effect of diferric Tf on TfR2. Anti-TfR1 antibody 42/6 was added to the medium of HepG2 cells at a concentration of 25 μg/mL 4 hours before the addition of 25 μM diferric Tf to the medium for 24 hours. To control for possible effects of iron deprivation, a second set of cells was treated identically, but 100 μM FeNTA was added concomitantly with 42/6 antibody. Lysates (20 μg total protein) were analyzed by Western blot. (B) TfR1 and TfR2 bands were visualized with fluorescence-labeled secondary antibodies. (C) Fluorescence imaging was used to quantitate TfR1 and TfR2 protein levels. The integrated intensity of each band is expressed as a percentage of control. The graph depicts the mean ± SD of 4 independent experiments. *P < .05, as determined by Student one-tailed paired t test.