Figure 1.
Figure 1. TfR2 increases after the addition of diferric Tf to the medium of HepG2 cells. (A) TfR2 increases in a time-dependent manner. HepG2 cells were cultured for 4 to 72 hours after the addition of 25 μM diferric Tf or HBS to the medium. Lysates (20 μg total protein) were transferred to nitrocellulose, probed for TfR2 and Tf, and visualized by chemiluminescence. The increase in TfR2 was paralleled by an increase in Tf associated with the cells. (B) TfR2 returns to basal levels after withdrawal of Tf from the medium. HepG2 cells were cultured for 24 hours in the presence of 25 μM diferric Tf, then chased in medium for 0 to 8 hours. TfR2 and Tf levels were analyzed by Western blot using HRP-conjugated secondary antibodies and chemiluminescence. (C) The response of TfR2 to diferric Tf is concentration dependent. HepG2 cells were cultured for 24 hours after the addition of 0 to 25 μM diferric Tf to the medium, and lysates (20 μg total protein) were analyzed by Western blot with fluorescence-labeled secondary antibodies for quantification and HRP-conjugated secondary antibodies for chemiluminescence imaging, as described in “Materials and methods.” The intensity of each band was normalized to the intensity of the 0 μM Tf sample. The log of the normalized intensity is plotted as a function of diferric Tf concentration. The increase in TfR2 is half-maximal at approximately 2.5 μM diferric Tf. (D) TfR2 does not increase in response to nontransferrin-bound iron. TfR2, TfR1, and Ft protein levels were assessed by Western blots of lysates (20 μg total protein) from HepG2 cells cultured for 24 hours in the presence of 100 μM FeNTA (lane 1,+) or 4 mM NTA (lane 2,-). Bands were detected by chemiluminescence. Ft heavy and light chains are visible as a doublet in the lower panel. (E) TfR2 does not increase in response to apo Tf. HepG2 cells were incubated in medium containing 25 μM diferric Tf (lane 1, labeled holo Tf) or apo Tf (lane 3) for 24 hours. Lysates (20 μg total protein) were analyzed by Western blot for TfR2, TfR1, and Ft protein. Bands were detected by chemiluminescence.

TfR2 increases after the addition of diferric Tf to the medium of HepG2 cells. (A) TfR2 increases in a time-dependent manner. HepG2 cells were cultured for 4 to 72 hours after the addition of 25 μM diferric Tf or HBS to the medium. Lysates (20 μg total protein) were transferred to nitrocellulose, probed for TfR2 and Tf, and visualized by chemiluminescence. The increase in TfR2 was paralleled by an increase in Tf associated with the cells. (B) TfR2 returns to basal levels after withdrawal of Tf from the medium. HepG2 cells were cultured for 24 hours in the presence of 25 μM diferric Tf, then chased in medium for 0 to 8 hours. TfR2 and Tf levels were analyzed by Western blot using HRP-conjugated secondary antibodies and chemiluminescence. (C) The response of TfR2 to diferric Tf is concentration dependent. HepG2 cells were cultured for 24 hours after the addition of 0 to 25 μM diferric Tf to the medium, and lysates (20 μg total protein) were analyzed by Western blot with fluorescence-labeled secondary antibodies for quantification and HRP-conjugated secondary antibodies for chemiluminescence imaging, as described in “Materials and methods.” The intensity of each band was normalized to the intensity of the 0 μM Tf sample. The log of the normalized intensity is plotted as a function of diferric Tf concentration. The increase in TfR2 is half-maximal at approximately 2.5 μM diferric Tf. (D) TfR2 does not increase in response to nontransferrin-bound iron. TfR2, TfR1, and Ft protein levels were assessed by Western blots of lysates (20 μg total protein) from HepG2 cells cultured for 24 hours in the presence of 100 μM FeNTA (lane 1,+) or 4 mM NTA (lane 2,-). Bands were detected by chemiluminescence. Ft heavy and light chains are visible as a doublet in the lower panel. (E) TfR2 does not increase in response to apo Tf. HepG2 cells were incubated in medium containing 25 μM diferric Tf (lane 1, labeled holo Tf) or apo Tf (lane 3) for 24 hours. Lysates (20 μg total protein) were analyzed by Western blot for TfR2, TfR1, and Ft protein. Bands were detected by chemiluminescence.

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