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1.
Figure 5

Figure 5. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphorylation of Y119 disrupts the interaction between Jak2 and EpoR. Jak2-deficient MEFs were coinfected with EpoR-FLAG and Jak2-HA mutants. Cells were stimulated with Epo (10 U/ml) for indicated periods. The cells were lysed and immunoprecipitated with anti-FLAG antibody and blotted with the antibodies anti-HA, phospho-Y1007/1008 Jak2, antiphospho tyrosine or anti-FLAG. Whole-cell lysates were immnoblotted with anti-HA antibody or anti-FLAG antibody.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
2.
Figure 6

Figure 6. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphorylation at Y119 reduces the stability of Jak2. Jak2-deficient MEFs were coinfected with EpoR and Jak2-HA mutants. Cells were pulse-labeled with 35S-methione for 40 min and chased for the indicated periods in the absence or presence of Epo (10 U/ml). Jak2 protein levels were analyzed by HA immunoprecipitation followed by autoradiographic exposure.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
3.
Figure 8

Figure 8. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphorylation at Y119 inhibits erythroid colony formation. (A, C) Wild type and Jak2-deficient fetal liver cells were subjected to in vitro colony assays with Epo and/or IL-3. (B, D) Fetal liver cells from Jak2-deficient embryos were infected with retrovirus encoding various Jak2 mutants and subjected to in vitro colony assays with Epo and/or IL-3. The benzidine-positive CFU-E colonies and BFU-E colonies were scored at day 3 and 8, respectively.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
4.
Figure 1

Figure 1. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Jak2 Y119, a conserved tyrosine, is phosphorylated in response to Epo. (A) Sequence alignment of Jak2 sequences from various species Jak family kinases. (B) MEFs were infected with EpoR and stimulated with Epo (10 U/ml) for indicated periods. Cell lysates were immunoblotted with antiphospho-Y119 Jak2 antibody, antiphospho-Y1007/1008 Jak2 antibody or anti-Jak2 antibody. (C) Jak2-deficient MEFs were coinfected with EpoR and Jak2-HA mutants. Cells were stimulated with Epo (10 U/ml) for 15 min. Cell lysates were subjected to immunoprecipitation using an anti-HA antibody and immunoblotted with antibodies to anti-phospho-Y119 Jak2, anti-phospho-Y1007/1008 Jak2, anti-phosphotyrosine or anti-Jak2.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
5.
Figure 2

Figure 2. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphopeptide mapping of Jak2. 293T cells were transfected with pRK5-Jak2 Flag, pRK5-Jak2 Y119F, pRK5-Jak2 Y124F or pRK5-Jak2 Y119F Y124F with MSCV-Puro EpoR. The cells were incubated with [32P]orthophosphate for 3 h and stimulated with Epo for 15 min. The lysates were immunoprecipitated with Flag antibody and eluted with Flag peptide. The eluted proteins were visualized by autoradiography (A). The digest of the eluted proteins was resolved in two dimensions on thin-layer cellulose plates with synthetic peptides (B). The 32P-labeled spots were visualized using a phosphoimager. The positions of synthetic peptides were determined by ninhydrin staining and are indicated by broken circles. The position of phosphopeptide(FYPFPHWYCSQSSR) is indicated by an arrow.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
6.
Figure 4

Figure 4. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphorylation of Y119 abrogates Epo-induced Jak2 phosphorylation at Y1007/Y1008 and STAT5 activation. Jak2-deficient MEFs were coinfected with EpoR and Jak2-HA mutants. Cells were stimulated with Epo (10 U/ml) for indicated periods. (A, B) The cell lysates were immunoprecipitated with anti-HA antibody and blotted with the antibodies antiphospho-Y1007/1008 Jak2, antiphosphotyrosine, phospho-Y119 Jak2 or anti-HA. (C) Cell lysates were immunoprecipitated with anti-Stat5 antibody and blotted with antiphospho tyrosine antibody (upper) or anti-Stat5 antibody (bottom). (D) Jak2-deficient MEFs were cotransfected with EpoR, Jak2 mutants and a Stat5-dependent luciferase reporter construct. Stat5 dependent luciferase activity was normalized to the activity of a constitutive, TK promoter driven Renilla luciferase construct.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
7.
Figure 7

Figure 7. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Receptor specificity of Y119 mutation. (A) Mouse embryonic fibroblasts were stimulated with IFNγ for the indicated times. Cell lysates were immunoblotted with anti-phospho-Y119 Jak2 antibody or with anti-Jak2 antibody. (B) Jak2-deficient fibroblasts were infected with wild type Jak2 or the indicated mutants. Cells were stimulated with IFNγ (5 ng/ml) for the indicated times. The cells were lysed and immunoprecipitated with anti-HA antibody. In vitro Jak2 kinase activity was measured by autophosphorylation of Jak2. Samples were separated by SDS–PAGE, transferred onto nitrocellulose and subjected to autoradiography (upper) or blotted with anti-HA antibody (bottom). (C) Cells were stimulated as above and cell lysates were immunoblotted and probed with the indicated antibodies to assess Jak2 activation (P-Y1007/1008; total tyrosine phosphorylation (PY), protein levels (HA) and activation of Stat1 (P-Stat1 Y701). (D) Jak2-deficient MEFs were coinfected with IFNγR2-FLAG and Jak2-HA mutants. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-HA antibody or anti-FLAG.

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.
8.
Figure 3

Figure 3. From: Receptor specific downregulation of cytokine signaling by autophosphorylation in the FERM domain of Jak2.

Phosphorylation of Y119 abrogates Epo-induced Jak2 activation. (A) Schematic structure of Jak2 mutants. The tyrosine residue at 119 was altered to phenylalanine (Y119F) or glutamic acid (Y119E). (B) Jak2-deficient MEFs were infected with Jak2-HA mutants. Cell lysates were immunoblotted with anti-HA antibody (upper panel) and anti-β-actin antibody (lower panel). (C) Jak2-deficient MEFs were coinfected with retroviruses encoding EpoR and Jak2-HA mutants. Cells were stimulated with Epo (10 U/ml) for indicated periods. The cells were lysed and immunoprecipitated with anti-HA antibody. In vitro kinase activity of Jak2 was measured by autophosphorylation of Jak2. Samples were then separated by SDS–PAGE, transferred to nitrocellulose, and subjected to autoradiography (upper panel) and blotting with anti-HA antibody (bottom panel). (D) In vitro kinase activity of Jak2 was measured in the presence of a synthetic peptide derived from the Y1007/Y1008 region of Jak2 (VLPQDKEYYKVKEPGES). Phosphorylated peptides were measured by scintillation counter (upper panel). Immunoprecipitated samples were separated by SDS–PAGE and subjected to blotting with anti-HA antibody (bottom panel).

Megumi Funakoshi-Tago, et al. EMBO J. 2006 October 18;25(20):4763-4772.

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