Figure 4. TfR2 stability is regulated by... | Rockefeller University Press

Figure 4.

$Figure 4. TfR2 stability is regulated by iron, isocitrate, and cathepsin activity. (A) Immunoblot of whole cell lysates from human progenitors cultured in iron-replete (100% TSAT) or -deficient (15% TSAT) erythroid medium ± isocitrate and treated with cycloheximide (CHX). (B) Densitometry for fraction of residual TfR2 at 3 versus 0 h of cycloheximide, with normalization to TfR1 (n = 3, one-way ANOVA). IC, isocitrate. (C) Immunoblot of membrane fractions from progenitors cultured in erythroid medium with indicated TSATs ± the cathepsin inhibitor CA074me. Representative results from three independent experiments (Fig. S4 A). (D) Plots from flow cytometry of progenitors cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. Graph depicts percent decrease in GPA expression associated with iron deprivation ± cathepsin inhibition (n = 3, Student’s t test). (E) IP of endogenous TfR1 and TfR2 using extracts from K562 cells untreated (Unt) or cultured with DFO or with FA followed by immunoblot detection. Right, input immunoblot. Representative results from three independent experiments. (F) IP of endogenous EpoR and TfR2 from extracts of HUDEP-2 cells cultured ± overnight iron withdrawal followed by immunoblot detection. Right, input immunoblot. Representative results from two independent experiments. IB, immunoblot. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.$

TfR2 stability is regulated by iron, isocitrate, and cathepsin activity. (A) Immunoblot of whole cell lysates from human progenitors cultured in iron-replete (100% TSAT) or -deficient (15% TSAT) erythroid medium ± isocitrate and treated with cycloheximide (CHX). (B) Densitometry for fraction of residual TfR2 at 3 versus 0 h of cycloheximide, with normalization to TfR1 (n = 3, one-way ANOVA). IC, isocitrate. (C) Immunoblot of membrane fractions from progenitors cultured in erythroid medium with indicated TSATs ± the cathepsin inhibitor CA074me. Representative results from three independent experiments (Fig. S4 A). (D) Plots from flow cytometry of progenitors cultured in erythroid medium with indicated TSATs ± cathepsin inhibitor. Graph depicts percent decrease in GPA expression associated with iron deprivation ± cathepsin inhibition (n = 3, Student’s t test). (E) IP of endogenous TfR1 and TfR2 using extracts from K562 cells untreated (Unt) or cultured with DFO or with FA followed by immunoblot detection. Right, input immunoblot. Representative results from three independent experiments. (F) IP of endogenous EpoR and TfR2 from extracts of HUDEP-2 cells cultured ± overnight iron withdrawal followed by immunoblot detection. Right, input immunoblot. Representative results from two independent experiments. IB, immunoblot. Graphs depict mean ± SEM from the indicated number of independent experiments. *, P < 0.05; ***, P < 0.001.