Journal: Leukemia

Article Title: Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.

doi: 10.1038/sj.leu.2402317

Figure Lengend Snippet: Figure 1 STAT5 expression and phosphorylation patterns of the investigated patients with AML. AML cells were cultured for 24 h in RPMI 1640 supplemented with 10% FBS. Total cell extracts were pre- pared and equal amounts of cell lysates were subjected to SDS-PAGE. Western blot analysis was performed using an antibody against phos- pho-STAT5. As a control, equal amounts of total cell lysates were Western blotted using antibodies against STAT5 A/B and actin. (Representative data from 15 cases are shown.)

Article Snippet: The proteins were electrophoretically transferred to PVDF membrane (Millipore, Bedford, MA, USA) and probed with polyclonal antibodies against STAT5 A/B (Upstate Biotechnology, Lake Placid, NY, USA), phosphorylated STAT5 or ERK (New England BioLabs, Beverly, MA, USA), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or monoclonal antibody against Bcl-xl (Zymed, South San Francisco, CA, USA) or actin (Boehringer, Mannheim, Germany).

Techniques: Expressing, Phospho-proteomics, Cell Culture, SDS Page, Western Blot, Control

Journal: Leukemia

Article Title: Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.

doi: 10.1038/sj.leu.2402317

Figure Lengend Snippet: Figure 2 Flt3 receptor is constitutively phosphorylated in majority of STAT5 positive AML cases. (a) 2 × 107 AML cells were cultured for 24 h in RPMI 1640 supplemented with 10% FBS. Cell lysates were prepared and Flt3 was immunoprecipitated from the lysates. After SDS-PAGE, Western blotting was performed using antibodies against phosphotyrosine (PY) or Flt3. (b) AML cells were cultured overnight in RPMI 1640 medium supplemented with 10% FBS and subsequently stimulated for 5 min with medium or FL (100 ng/ml). Cell lysates were prepared and Flt3 was immunoprecipitated from the lysates. After SDS-PAGE, Western blotting was performed using antibodies against phosphotyrosine (PY) or Flt3. (c) AML cells (case 23) were cultured overnight in RPMI 1640 supplemented with 10% FBS. Subsequently cells were stimulated for 10 min with the indicated AML supernatants or FL as a positive control. Total cell extracts were prepared and sub- jected to SDS-PAGE. Western blotting was performed using antibodies against phospho-STAT5 and STAT5 A/B. (d) AML cells were cultured overnight in RPMI 1640 medium with 10% FBS with or without anti- GM-CSF (1 g/ml). Total cell extracts were prepared and subjected to SDS-PAGE. Western blotting was performed using antibodies against phospho-STAT5 and STAT5 A/B.

Article Snippet: The proteins were electrophoretically transferred to PVDF membrane (Millipore, Bedford, MA, USA) and probed with polyclonal antibodies against STAT5 A/B (Upstate Biotechnology, Lake Placid, NY, USA), phosphorylated STAT5 or ERK (New England BioLabs, Beverly, MA, USA), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or monoclonal antibody against Bcl-xl (Zymed, South San Francisco, CA, USA) or actin (Boehringer, Mannheim, Germany).

Techniques: Cell Culture, Immunoprecipitation, SDS Page, Western Blot, Positive Control

Journal: Leukemia

Article Title: Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.

doi: 10.1038/sj.leu.2402317

Figure Lengend Snippet: Figure 3 Specific tyrosine kinase III inhibitor AG1296 inhibits FL- induced STAT5 and ERK phosphorylation. (a) AML cells were cultured overnight in RPMI 1640 supplemented with 10% FBS. Subsequently cells were pre-incubated for 90 min with AG1296 (20 M) and then stimulated for 10 min with FL (100 ng/ml). Total cell extracts were prepared and subjected to SDS-PAGE. Western blotting was perfor- med using antibodies against phospho-STAT5, STAT5 A/B, phospho- ERK1/2 and ERK1/2. (b) AML cells were cultured overnight in RPMI 1640 supplemented with 10% FBS. Subsequently cells were pre-incu- bated for 90 min with AG1296 (20 M) or AG490 (100 M) and then stimulated for 10 min with FL (100 ng/ml) or GM-CSF (10 ng/ml). Total cell extracts were prepared and subjected to SDS-PAGE. West- ern blotting was performed using antibodies against phospho-STAT5 and STAT5 A/B. (c) AML cells were cultured overnight in RPMI 1640 supplemented with 10% FBS. Subsequently cells were pre-incubated for 90 min with AG1296 (20 M) and then stimulated for 5 min with FL (100 ng/ml). Cell lysates were prepared and Flt3 was immunopreci- pitated from the lysates. After SDS-PAGE, Western blotting was perfor- med using antibodies against phosphotyrosine (PY) or Flt3.

Article Snippet: The proteins were electrophoretically transferred to PVDF membrane (Millipore, Bedford, MA, USA) and probed with polyclonal antibodies against STAT5 A/B (Upstate Biotechnology, Lake Placid, NY, USA), phosphorylated STAT5 or ERK (New England BioLabs, Beverly, MA, USA), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or monoclonal antibody against Bcl-xl (Zymed, South San Francisco, CA, USA) or actin (Boehringer, Mannheim, Germany).

Techniques: Phospho-proteomics, Cell Culture, Incubation, SDS Page, Western Blot

Journal: Leukemia

Article Title: Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts.

doi: 10.1038/sj.leu.2402317

Figure Lengend Snippet: Figure 4 Constitutive Flt3 and STAT5 phosphorylation is inhibited by AG1296. (a) AML cells were cultured overnight in RPMI 1640 sup- plemented with 10% FBS with or without AG1296 (20 M). Cell lys- ates were prepared and Flt3 was immunoprecipitated from the lysates. After SDS-PAGE, Western blotting was performed using antibodies against phosphotyrosine (PY) or Flt3. (b) AML cells were cultured over- night in RPMI 1640 supplemented with 10% FBS with or without AG1296 (20 M). Total cell extracts were prepared and equal amounts of cell lysates were subjected to SDS-PAGE. Western blot analysis was performed using antibodies against phospho-STAT5 or STAT5 A/B.

Article Snippet: The proteins were electrophoretically transferred to PVDF membrane (Millipore, Bedford, MA, USA) and probed with polyclonal antibodies against STAT5 A/B (Upstate Biotechnology, Lake Placid, NY, USA), phosphorylated STAT5 or ERK (New England BioLabs, Beverly, MA, USA), ERK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or monoclonal antibody against Bcl-xl (Zymed, South San Francisco, CA, USA) or actin (Boehringer, Mannheim, Germany).

Techniques: Phospho-proteomics, Cell Culture, Immunoprecipitation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: A) Scatchard's plot analysis: Effects of anti-IL-15Rβ and γc mAbs on IL-15 binding to RCC. For the IL-15 binding experiments, RCC7 cells were incubated with increasing concentrations of radioiodinated rIL-15 in presence or not of the following neutralizing mAbs: anti-IL-2Rβ and IL-2Rγ. The nonspecific cell binding was determined in the presence of radioiodinated rhIL-15 and a 100-fold excess of unlabeled rhIL-15. Cell-bound (B) and unbound (free, F) fractions were measured, and the specific bound fraction was calculated by subtracting the nonspecific binding from the cell-bound fraction. On the ordinate is plotted the ratio of the specific bound fraction (expressed in sites per cell) over the total concentration (bound plus free) of radioiodinated rIL-15 (expressed in pM). On the abscissa bound fraction (expressed in sites per cell). The high affinity specific IL-15 binding (Kd = 375 pM, 413 IL-15 binding sites per cell), which was completely abrogated by neutralizing antibody against the IL-2Rβ (inset) but not the γc chain, suggested the presence on RCC of an IL-15Rα/IL-2Rβ complex. B ) Detection of IL-15Rαβ complex by immunoprecipitation (IP) with anti-IL-15Rα (M161) or mouse IgG protein G-Sepharose-conjugate on total lysate (TL) of RCC7. Immunoprecipitated complexes were blotted either with anti-IL-2Rβ (sc-1046) and anti-IL-15Rα (sc-9172). C ) Stimulation for 10 and 40 min with physiologic (10 pg/mL) and supra-physiologic (10 ng/mL) concentrations of rhIL-15 induces the phosphorylation of MAPK ERK1/2 and IκBα in RPTEC and RCC7, whereas STAT5 activation was only observed in RPTEC. Histograms represent densitometry comparison of each factor normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) and 3 RPTEC batches. * P<0.05 versus control, Mann-Whitney test. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Binding Assay, Incubation, Concentration Assay, Immunoprecipitation, Phospho-proteomics, Activation Assay, Comparison, Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Neutralization, Inhibition, Expressing, Recombinant, Control, Fluorescence, Marker

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. A ) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. B ) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. C ) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Transfection, Expressing, Western Blot, Control, Flow Cytometry, Phospho-proteomics, Fluorescence, Marker