Figure 2.
Figure 2. JAK2 inhibition does not affect hepcidin-mediated FPN1 degradation in vitro or in vivo. (A) Representative western blot and quantification (n = 3 per group) of FPN1 protein levels in murine macrophage RAW 264.7 cells. Control-treated (ctrl) RAWs were not treated at all. All other groups were stimulated with FeCl3 (50 µM) for 20 hours to induce FPN1 expression. Where indicated, cells were further treated with hepcidin (Hep; 1 µg/mL) alone or together with increasing concentrations of MMB (10 nM, 100 nM, 500 nM, and 1 µM) for an additional 6 hours. β-actin levels were used for normalization. Each lane represents one individual replicate. (B) Western blot of JAK2 protein levels in bone marrow–derived macrophages (BMDMs) from JAK2Wt and JAK2cKO mice. β-actin levels were used as a loading control (top). Western blot of pSTAT5 protein levels in BMDMs from JAK2Wt and JAK2cKO mice after stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF; 100 ng/mL) for 30 minutes (bottom). STAT5 levels were used as a loading control. Each lane represents one individual animal. (C) Representative western blots and quantification (n = 6-9 per group) of FPN1 protein levels in BMDMs from JAK2Wt and JAK2cKO mice incubated with either FeCl3 (50 µM) for 20 hours alone or with hepcidin (1 µg/mL) for 6 hours. Ctrl BMDMs received neither FeCl3 nor hepcidin. β-actin levels were used for normalization. Each lane represents one individual animal. (D-E) Representative western blot and quantification (n = 5-7) of FPN1 protein levels in whole-spleen lysates (D) and serum iron levels (E) from JAK2Wt and JAK2cKO mice at baseline (ctrl) and after intraperitoneal administration of hepcidin (4 mg/kg) for 3 hours. β-actin levels were used for normalization. Each lane represents one individual animal (D). Analysis of variance with Dunnett’s correction for multiple comparisons vs iron (Fe)/Hep-stimulated cells was applied (A,C). Unpaired Student t test was applied for comparison in each group (D-E). Results are shown as means ± standard errors of the mean. ***P < .001, ****P < .0001.

JAK2 inhibition does not affect hepcidin-mediated FPN1 degradation in vitro or in vivo. (A) Representative western blot and quantification (n = 3 per group) of FPN1 protein levels in murine macrophage RAW 264.7 cells. Control-treated (ctrl) RAWs were not treated at all. All other groups were stimulated with FeCl3 (50 µM) for 20 hours to induce FPN1 expression. Where indicated, cells were further treated with hepcidin (Hep; 1 µg/mL) alone or together with increasing concentrations of MMB (10 nM, 100 nM, 500 nM, and 1 µM) for an additional 6 hours. β-actin levels were used for normalization. Each lane represents one individual replicate. (B) Western blot of JAK2 protein levels in bone marrow–derived macrophages (BMDMs) from JAK2Wt and JAK2cKO mice. β-actin levels were used as a loading control (top). Western blot of pSTAT5 protein levels in BMDMs from JAK2Wt and JAK2cKO mice after stimulation with granulocyte macrophage colony-stimulating factor (GM-CSF; 100 ng/mL) for 30 minutes (bottom). STAT5 levels were used as a loading control. Each lane represents one individual animal. (C) Representative western blots and quantification (n = 6-9 per group) of FPN1 protein levels in BMDMs from JAK2Wt and JAK2cKO mice incubated with either FeCl3 (50 µM) for 20 hours alone or with hepcidin (1 µg/mL) for 6 hours. Ctrl BMDMs received neither FeCl3 nor hepcidin. β-actin levels were used for normalization. Each lane represents one individual animal. (D-E) Representative western blot and quantification (n = 5-7) of FPN1 protein levels in whole-spleen lysates (D) and serum iron levels (E) from JAK2Wt and JAK2cKO mice at baseline (ctrl) and after intraperitoneal administration of hepcidin (4 mg/kg) for 3 hours. β-actin levels were used for normalization. Each lane represents one individual animal (D). Analysis of variance with Dunnett’s correction for multiple comparisons vs iron (Fe)/Hep-stimulated cells was applied (A,C). Unpaired Student t test was applied for comparison in each group (D-E). Results are shown as means ± standard errors of the mean. ***P < .001, ****P < .0001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal