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1.
FIGURE 7.

FIGURE 7. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Co-immunoprecipitation shows evidence of IL-9Rα homodimerization. HEK293 cells were transiently co-transfected with Myc- and/or HA-tagged IL-9Rα or HA-tagged EPOR. 24 h post-transfection, cellular extracts were immunoprecipitated (IP) with an anti-Myc antibody and analyzed by Western blot with anti-HA antibodies to detect co-immunoprecipitation of IL-9Rα. Anti-Myc antibodies were used as the control, and total lysates were analyzed with the same antibodies. Similar results were obtained in three independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
2.
FIGURE 6.

FIGURE 6. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

IL-9Rα BOX1 mutant, but not TPOR or EPOR, inhibits IL-9Rα-mediated activation of STAT3 by JAK1 V658F. HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F, IL-9Rα wild-type, and/or BOX1 mutant or/and with the thrombopoietin or erythropoietin receptors in addition to the STAT3-responsive luciferase reporter. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in two independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
3.
FIGURE 8.

FIGURE 8. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Both IL-9Rα and IL-2Rß allows for JAK1 V658F- and JAK1 A634D-induced constitutive activation of STAT5. A, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type, V658F or A634D, IL-9Rα and/or γc in addition to the STAT5-responsive luciferase reporter pLHRE. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, the same experiment was performed with IL-2Rβ. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
4.
FIGURE 1.

FIGURE 1. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

ALL-associated JAK1 mutants (V658F and A634D) induce constitutive STAT3 activation in the presence of IL-9Rα. HEK293 cells were transiently transfected with different JAK1 constructs alone or in combination with IL-9Rα or the mutated IL-9Rα Y116F. The STAT3-responsive pGL3-pap1 construct was used as luciferase reporter. 4 h post-transfection, cells were incubated for 20 h with or without IL-9 before the luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
5.
FIGURE 4.

FIGURE 4. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Co-expression of JAK3 partially abrogates the γc inhibitory effect on IL-9Rα-mediated STAT3 activation by JAK1 V658F. HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F, γc, IL-9Rα, and/or with JAK3 as the last component of IL-9R complex in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 4 h post-transfection cells were incubated for 20 h with or without IL-9 and subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
6.
FIGURE 2.

FIGURE 2. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Y107A mutation in the FERM domain of JAK1 mutants abolishes IL-9Rα-mediated constitutive STAT3 activation. COS-7 (A) or HEK293 (B) cells were transiently co-transfected with empty vector or different JAK1 constructs with intact or mutated Y107A FERM domain (V658F/Y107A or A634D/Y107A) and with or without IL-9Rα. The STAT3-responsive pGL3-pap1 construct was used as luciferase reporter. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments in COS-7 and HEK293 cells.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
7.
FIGURE 5.

FIGURE 5. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Dominant negative effect of IL-9Rα BOX1 mutant on constitutive JAK1 V658F phosphorylation and STAT3 activation mediated by IL-9Rα WT. A, HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F and different IL-9Rα constructs in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, HEK293 cells were transiently co-transfected with JAK1 wild-type or V658F and different IL-9Rα constructs. 24 h post-transfection 106 cells were lysed and subjected to Western blot analysis. Phosphorylation of JAK1 and STAT3 was detected using specific anti-pJAK1 Tyr-1022/1023 and anti-pSTAT3 Tyr-705 antibodies. Membranes were reprobed with anti-JAK1, anti-STAT3, and anti-β-actin antibodies as control. Similar results were obtained in three independent experiments.

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.
8.
FIGURE 3.

FIGURE 3. From: Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor ? Homodimers.

Co-expression of γc inhibits IL-9Rα-mediated JAK1 phosphorylation and STAT3 activation. A, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type or V658F, γc, or/and IL-9Rα in addition to the STAT3-responsive luciferase reporter pGL3-pap1. 24 h post-transfection cells were subjected to a luciferase assay. Results are the mean ± variation of duplicate samples. Similar results were obtained in three independent experiments. B, HEK293 cells were transiently co-transfected with different empty vector, JAK1 constructs, γc, or/and IL-9Rα. 24 h post-transfection, 106 cells were lysed and subjected to Western blot analysis. Phosphorylation of JAK1 and STAT3 was detected using specific anti-pJAK1 Tyr-1022/1023 and anti-pSTAT3 Tyr-705 antibodies. Membranes were reprobed with anti-JAK1, anti-STAT3 and anti-β-actin antibodies as control. Similar results were obtained in two independent experiments. C, HEK293 cells were transiently co-transfected with empty vector, JAK1 wild-type or V658F, and IL-9Rα with or without γc in addition to the STAT3-responsive luciferase reporter pGL3-pap1. One day post-transfection, an aliquot of cells was used to assess cell surface expression of IL-9Rα by FACS analysis using anti-human IL-9Rα antibody followed with phycoerythrin-conjugated streptavidin (SAPE).

Tekla Hornakova, et al. J Biol Chem. 2009 March 13;284(11):6773-6781.

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