Figure 1.

Fpn-GFP is ubiquitinated and phosphorylated. (A) HEK293T cells (stably transfected with an inducible Fpn-GFP) induced to express Fpn-GFP (+ponasterone) were incubated in the presence or absence of 1 μg/ml hepcidin for 15 min. Cells were placed at 0°C and solubilized in 1.0% Triton X-100, 150 mM NaCl, 10 mM EDTA, 10 mM Tris, pH 7.4, protease inhibitor mixture, 50 mM N-ethylmaleimide, and protein phosphatase inhibitor set. Samples were immunoprecipitated with rabbit anti-GFP antibodies as described in Materials and Methods. Immunoprecipitated samples were analyzed by Western blots probing for ubiquitin by using mouse anti-ubiquitin or for phosphotyrosine by using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG as secondary antibody. Blots were also probed for Fpn-GFP by using rabbit anti-GFP followed by peroxidase-conjugated goat anti-rabbit IgG as secondary. (B) HEK293T cells were induced to express Fpn-GFP with ponasterone for 18 h. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for the indicated times, placed at 0°C, and the cell surface was biotinylated using sulfo-NHS-SS-biotin. After biotinylation, cells were solubilized as described in A, and the biotinylated proteins were affinity purified using streptavidin affinity gel. The affinity-purified samples were analyzed by Western blot using a rabbit anti-GFP followed by peroxidase-conjugated goat anti-rabbit IgG. (C) HEK293T Fpn-GFP cells were induced to express Fpn-GFP as described in B. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 15 min, placed at 0°C, and the cell surface was biotinylated. Biotinylated samples were purified using streptavidin beads as described in B. Affinity-purified samples were analyzed by Western blot with mouse anti-phosphotyrosine, mouse anti-ubiquitin followed by a peroxidase-conjugated goat anti-mouse IgG as secondary, or Fpn-GFP by using rabbit anti-GFP followed by peroxidase-conjugated goat anti-rabbit IgG as secondary. “Control” in the top panel is a mixture of tyrosine-modified proteins purchased from Calbiochem as a control for the anti-phosphotyrosine antibody. Flow-through is the sample that did not bind the streptavidin affinity gel (nonbiotinylated Fpn-GFP) but that was subsequently immunopurified using rabbit anti-GFP and the immunopurified sample analyzed by Western blot to test for the presence of phosphotyrosine or ubiquitin modifications on Fpn-GFP.