Figure 4
Figure 4. TpoR down-regulation induced by JAK2 V617F is promoted by proteasome degradation through stronger receptor ubiquitinylation in cell lines and in platelets from JAK2 V617F knock-in mice. In vivo proteasome inhibitor treatment restored TpoR levels. (A) The indicated Ba/F3 cell lines were lysed in 1% NP40 buffer, immunoprecipitated with anti-HA, and subjected to Western blotting with anti-HA antibodies or anti–polyubiquitin/monoubiquitin antibodies. Vertical line(s) have been inserted to indicate a repositioned gel lane. (B) HEK293-derived BOSC cells were transfected with the indicated TpoR and JAK2 constructs and with or without Flag-tagged ubiquitin. Two days after transfection, cells were treated with biotinylated Tpo (bio/Tpo) for 5 minutes at room temperature and 15 minutes at 37°C. Cells were lysed in 1% NP40 buffer, incubated streptavidin beads for 1 hour at 4°C. Beads were extensively washed and lysed in Laemmli buffer. Western blot was performed with anti-HA, anti-Flag, and anti–β-actin antibodies. In parallel, total cell lysates of the transfected cells were analyzed by Western blotting. (C) Left panel: Schematic representation of the TpoR cytosolic region that contains the 2 lysine K40 and K60 residues potentially involved in ubiquitinylation and the 2 leucine L54 and L55 residues involved in internalization. Right panel: HEK293-derived BOSC cells were transfected with the indicated TpoR constructs and Flag-tagged ubiquitin or empty vector. Two days after transfection, cells were stimulated with 50 ng/mL Tpo for 20 minutes. Cells were lysed in Laemmli buffer and examined by Western blotting with anti-HA, anti-Flag (for Flag-ubiquitin), or anti–β-actin. (D) Western blot analysis of platelet extracts from the indicated mice with anti-Mpl (TpoR; UBI) and anti–β-actin antibodies. JAK2wt-1 and JAK2wt-2 mice are wild-type C57Bl6 mice, KI/JAK2V617F-6 and -7 are heterozygous JAK2 V617F knock-in mice, JAK2wt-3 and JAK2wt-4 are C57Bl6 mice reconstituted with bone marrow from wild-type mice, whereas KI/JAK2V617F-7 and -8 are mice reconstituted with bone marrow from heterozygous JAK2 V617F knock-in mice. (E) Western blot analysis of paired platelet samples collected before and after drug treatment from the same KI-JAK2 V617F mouse. Mice were treated either with vehicle control (KI-JAK2 V617F-13) or bortezomib (0.5 mg/kg; KI-JAK2 V617F-14) during 2 weeks by intraperitoneal injections. (F) Western blot analysis of platelet extracts from the indicated mice that were reconstituted with bone marrow from JAK2 V617F KI mice. Mice were treated either with vehicle (KI/JAK2 V617F-9) or bortezomib (KI/JAK2 V617F-10, -11, and -12). Mice were treated for 2 weeks (twice per week) with intraperitoneal injections of the proteasome inhibitor bortezomib (0.5 mg/kg).

TpoR down-regulation induced by JAK2 V617F is promoted by proteasome degradation through stronger receptor ubiquitinylation in cell lines and in platelets from JAK2 V617F knock-in mice. In vivo proteasome inhibitor treatment restored TpoR levels. (A) The indicated Ba/F3 cell lines were lysed in 1% NP40 buffer, immunoprecipitated with anti-HA, and subjected to Western blotting with anti-HA antibodies or anti–polyubiquitin/monoubiquitin antibodies. Vertical line(s) have been inserted to indicate a repositioned gel lane. (B) HEK293-derived BOSC cells were transfected with the indicated TpoR and JAK2 constructs and with or without Flag-tagged ubiquitin. Two days after transfection, cells were treated with biotinylated Tpo (bio/Tpo) for 5 minutes at room temperature and 15 minutes at 37°C. Cells were lysed in 1% NP40 buffer, incubated streptavidin beads for 1 hour at 4°C. Beads were extensively washed and lysed in Laemmli buffer. Western blot was performed with anti-HA, anti-Flag, and anti–β-actin antibodies. In parallel, total cell lysates of the transfected cells were analyzed by Western blotting. (C) Left panel: Schematic representation of the TpoR cytosolic region that contains the 2 lysine K40 and K60 residues potentially involved in ubiquitinylation and the 2 leucine L54 and L55 residues involved in internalization. Right panel: HEK293-derived BOSC cells were transfected with the indicated TpoR constructs and Flag-tagged ubiquitin or empty vector. Two days after transfection, cells were stimulated with 50 ng/mL Tpo for 20 minutes. Cells were lysed in Laemmli buffer and examined by Western blotting with anti-HA, anti-Flag (for Flag-ubiquitin), or anti–β-actin. (D) Western blot analysis of platelet extracts from the indicated mice with anti-Mpl (TpoR; UBI) and anti–β-actin antibodies. JAK2wt-1 and JAK2wt-2 mice are wild-type C57Bl6 mice, KI/JAK2V617F-6 and -7 are heterozygous JAK2 V617F knock-in mice, JAK2wt-3 and JAK2wt-4 are C57Bl6 mice reconstituted with bone marrow from wild-type mice, whereas KI/JAK2V617F-7 and -8 are mice reconstituted with bone marrow from heterozygous JAK2 V617F knock-in mice. (E) Western blot analysis of paired platelet samples collected before and after drug treatment from the same KI-JAK2 V617F mouse. Mice were treated either with vehicle control (KI-JAK2 V617F-13) or bortezomib (0.5 mg/kg; KI-JAK2 V617F-14) during 2 weeks by intraperitoneal injections. (F) Western blot analysis of platelet extracts from the indicated mice that were reconstituted with bone marrow from JAK2 V617F KI mice. Mice were treated either with vehicle (KI/JAK2 V617F-9) or bortezomib (KI/JAK2 V617F-10, -11, and -12). Mice were treated for 2 weeks (twice per week) with intraperitoneal injections of the proteasome inhibitor bortezomib (0.5 mg/kg).

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