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Results: 4

1.
Figure 2

Figure 2. From: The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

Direct association of TfR with HFE. (A) HFE antibodies coimmunoprecipitate TfR from wild-type and H63D HFE expressing cells but very little from parental 293 or C282Y HFE mutant expressing cells. (B) HFE antibodies immunoprecipitate substantial amounts of HFE protein from wild-type, C282Y and H63D HFE expressing cells. (C) TfR antibodies coimmunoprecipitate HFE from wild-type and H63D HFE expressor cells but not parental 293 and only a trace from C282Y mutant expressing cells. (D) TfR antibodies immunoprecipitate TfR protein from parental 293, and wild-type, C282Y and H63D HFE expressing cells. (E) FLAG epitope (M2) antibodies coimmunoprecipitate TfR from wild-type and H63D HFE expressing cells but not parental 293 or C282Y HFE mutant expressing cells.

John N. Feder, et al. Proc Natl Acad Sci U S A. 1998 February 17;95(4):1472-1477.
2.
Figure 1

Figure 1. From: The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

Cell-surface labeling of HFE and association with TfR. (A) HFE antibodies immunoprecipitate 12, 49, 100, and 200 kDa surface-labeled proteins from wild-type HFE expressing cells. The coimmunoprecipitating proteins are greatly reduced or absent in immune complexes from parental 293 or C282Y HFE mutant expressing cells. (B) FLAG epitope antibodies also immunoprecipitate 12, 49, 100, and 200 kDa surface-labeled proteins in wild-type HFE expressing cells. As in A, the coimmunoprecipitating proteins are greatly reduced or absent in immune complexes from parental 293 or C282Y HFE mutant expressing cells. (C) TfR antibodies immunoprecipitate 100- and 200-kDa surface-labeled proteins from parental 293, wild-type, and C282Y HFE expressing cells and in addition, immunoprecipitate 12- and 49-kDa proteins from wild-type HFE expressing cells. (D) HLA-ABC antibodies fail to immunoprecipitate 100- and 200-kDa proteins from parental 293 cells.

John N. Feder, et al. Proc Natl Acad Sci U S A. 1998 February 17;95(4):1472-1477.
3.
Figure 3

Figure 3. From: The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

Effect of HFE on cell-association of [125I]-transferrin. (A) Cell association of [125I]-transferrin in cells that overexpress the C282Y (intracellular) mutant form of HFE. Approximately 9 × 105 cells (clone 10, □; and clone 12, ▪) were incubated with various concentrations of transferrin at 37°C for 20 min (Inset). The data represent the mean of duplicate determinations corrected for nonspecific binding. Scatchard analysis revealed an Kcell association of ≈12 and 14 nM, respectively. (B) Cell-association of [125I]-transferrin in cells overexpressing the wild-type (surface) form of HFE (clone 7, ○; clone 3, •). Scatchard analysis revealed that the Kcell association for transferrin was 180 and 40 nM, respectively. (C) Cell-association of [125I]-transferrin in cells overexpressing the H63D mutant form of HFE (clone 3, ▵; clone 8, ▴). Scatchard analysis revealed a Kcell association for transferrin to be 12 and 20 nM, respectively. (D) Inhibition of cell-associated [125I]-transferrin to the TfR on parental 293 cells by addition of soluble HFE/β2m heterodimers. Parental 293 cells were preincubated with various amounts of soluble HFE/β2m heterodimers followed by addition of 10 nM [125I]-transferrin. The amount of specific cell-associated transferrin was determined and plotted as a percent of that observed in cells with no added HFE. The apparent Ki is 87 nM.

John N. Feder, et al. Proc Natl Acad Sci U S A. 1998 February 17;95(4):1472-1477.
4.
Figure 4

Figure 4. From: The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.

Soluble HFE/β2m heterodimers reduce the affinity of the TfR for transferrin. (A) Effect of HFE/β2m heterodimers on the cell-association of [125I]-transferrin to HeLa cells at 37°C. Cell-associated [125I]-transferrin in HeLa cells in the presence of 0, 150, or 1,000 nM HFE or 100 nM soluble HLA-B27/β2m heterodimers (Inset). Scatchard analysis of cell associated [125I]-transferrin in the presence of 0 nM (□), 150 nM (▴), or 1,000 nM HFE (▵) revealed Kcell association values of 4.7 nM, 11 nM, and 50 nM, respectively and ≈1.0 × 106 transferrin binding sites per cell. (Note: the addition of 1,000 nM soluble HFE/β2m heterodimers dramatically reduces cell-associated transferrin such that an accurate x-intercept is difficult to obtain; the Kcell association 50 nM is estimated utilizing 1.0 × 106 binding sites per cell.) Addition of 100 nM soluble HLA-B27/β2m heterodimers (▪) to the assay had no effect. (B) Determination of the apparent Ki of soluble HFE/β2m heterodimers for cell-associated transferrin inhibition in HeLa cells. Cells were preincubated at 37°C in various amounts of soluble HFE/β2m heterodimers and then [125I]-transferrin was added to 5 nM. The amount of specific cell-associated transferrin was determined and plotted as a percent of that observed in cells with no added HFE. The apparent Ki for HeLa is 39 nM. (C) Effect of HFE/β2m heterodimers on the binding of [125I]-transferrin to HeLa cells at 4°C. Binding of [125I]-transferrin to HeLa cells in the absence or presence of 0, 35, or 1,000 nM HFE (inset). Scatchard analysis of [125I]-transferrin binding in the presence of 0 (□), 35 (▴), or 1,000 nM (▵) HFE revealed apparent KD values of 5 nM, 12 nM, and 75 nM, respectively and ≈1.0 × 106 transferrin binding sites per cell. (Note: the addition of 1,000 nM soluble HFE/β2m heterodimers dramatically reduces transferrin binding such that an accurate x intercept is difficult to obtain; the apparent KD of 75 nM is estimated utilizing 1.0 × 106 binding sites per cell.)

John N. Feder, et al. Proc Natl Acad Sci U S A. 1998 February 17;95(4):1472-1477.

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