Figure 2
Figure 2. SH2 domain-mutated JAK2V617F can be rescued by forced overexpression of EpoR. (A) JAK2V617F-expressing Ba/F3 (V617F) and EpoR-Ba/F3 (EpoR) cells were selected by IL-3 withdrawal, serum-starved for 12 hours and stimulated with IL-3, Epo, or vehicle. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) EpoR-Ba/F3 cells expressing WT-JAK2 and the indicated JAK2 mutants described in Figure 1A were grown in the absence of Epo. Cell growth was measured by MTS-based assay and by trypan blue exclusion as in Figure 1A. The figures represent 1 of 3 independent experiments. Values are expressed as mean of triplicates ± SEM. (C) EpoR-Ba/F3 cells expressing WT or mutant JAK2 as indicated and selected by IL-3 withdrawal as described in panel B were serum-starved for 12 hours and stimulated with Epo or vehicle for 5 minutes. Lysates were subjected to Western blotting with the indicated antibodies. Note that the detection of JAK2V617F mFERM required longer exposures compared with the other JAK2 constructs. (D) EpoR-Ba/F3 cells containing WT-JAK2 or the JAK2 mutants as indicated were serum-starved and stimulated with Epo or vehicle. Flag-tagged JAK2 was IP from whole-cell lysates by an anti-Flag antibody and immuoblotted with the indicated antibodies. (E) Ba/F3 JAK2V617F mSH2 cells were transduced with MSCV-eGFP-EpoR (EpoR+V617F mSH2) and grown in the presence or absence of IL-3 over a period of 3 days. FACS analysis was used to determine surface expression levels of EpoR (left panel). Analysis of JAK2 and STAT5 activation in these 2 EpoR-expressing Ba/F3 cell populations in the absence and presence of Epo was determined by Western blot (right panel). (F) Proliferation of isolated SH2-mutated JAK2V617F (V617F mSH2) Ba/F3 single-cell clones expressing high or low levels of EpoR as quantified by the relative optical density (OD) using MTS assay. The figure represents 1 of 2 independent experiments. Values are expressed as mean of triplicates ± SEM (left panel). EpoR expression levels were determined by Western blot analysis (right panel).

SH2 domain-mutated JAK2V617F can be rescued by forced overexpression of EpoR. (A) JAK2V617F-expressing Ba/F3 (V617F) and EpoR-Ba/F3 (EpoR) cells were selected by IL-3 withdrawal, serum-starved for 12 hours and stimulated with IL-3, Epo, or vehicle. Cell lysates were subjected to immunoblotting using the indicated antibodies. (B) EpoR-Ba/F3 cells expressing WT-JAK2 and the indicated JAK2 mutants described in Figure 1A were grown in the absence of Epo. Cell growth was measured by MTS-based assay and by trypan blue exclusion as in Figure 1A. The figures represent 1 of 3 independent experiments. Values are expressed as mean of triplicates ± SEM. (C) EpoR-Ba/F3 cells expressing WT or mutant JAK2 as indicated and selected by IL-3 withdrawal as described in panel B were serum-starved for 12 hours and stimulated with Epo or vehicle for 5 minutes. Lysates were subjected to Western blotting with the indicated antibodies. Note that the detection of JAK2V617F mFERM required longer exposures compared with the other JAK2 constructs. (D) EpoR-Ba/F3 cells containing WT-JAK2 or the JAK2 mutants as indicated were serum-starved and stimulated with Epo or vehicle. Flag-tagged JAK2 was IP from whole-cell lysates by an anti-Flag antibody and immuoblotted with the indicated antibodies. (E) Ba/F3 JAK2V617F mSH2 cells were transduced with MSCV-eGFP-EpoR (EpoR+V617F mSH2) and grown in the presence or absence of IL-3 over a period of 3 days. FACS analysis was used to determine surface expression levels of EpoR (left panel). Analysis of JAK2 and STAT5 activation in these 2 EpoR-expressing Ba/F3 cell populations in the absence and presence of Epo was determined by Western blot (right panel). (F) Proliferation of isolated SH2-mutated JAK2V617F (V617F mSH2) Ba/F3 single-cell clones expressing high or low levels of EpoR as quantified by the relative optical density (OD) using MTS assay. The figure represents 1 of 2 independent experiments. Values are expressed as mean of triplicates ± SEM (left panel). EpoR expression levels were determined by Western blot analysis (right panel).

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