Skip to main content
. 2009 Feb 20;106(10):3800–3805. doi: 10.1073/pnas.0900453106

Fig. 6.

Fig. 6.

Hepcidin-mediated activation of Fpn bound Jak2 is highly cooperative. (A) HEK293T cells were transiently transfected with WT Fpn-FLAG and Fpn(Y302–303F)-GFP expressed under a CMV promoter. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-GFP or anti-FLAG antibodies as in Fig. 5. Immunoprecipitated samples were analyzed by Western blot using mouse anti-phosphotyrosine (1) or rabbit anti-Jak2 (2) followed by a peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. A second immunoprecipitation was performed as in (A) and Jak2 phosphorylation assessed using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (panel 3). (B) HEK293T cells were transiently transfected with WT Fpn-FLAG and Fpn(N144H)-GFP expressed under a CMV promoter. Cells were incubated in the presence or absence of 1 μg/ml hepcidin for 30 min, solubilized, and immunoprecipitated with rabbit anti-GFP or anti-FLAG antibodies. Immunoprecipitated samples were analyzed by Western blots using mouse anti-phosphotyrosine (1) or rabbit anti-Jak2 (2) followed by a peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG. A second immunoprecipitation was performed as in (A) and Jak2 phosphorylation assessed using mouse anti-phosphotyrosine followed by a peroxidase-conjugated goat anti-mouse IgG (3).