Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Clinical information and Stat5 activation status of primary human samples Pre-existing, remnant primary human leukemia, and lymphoma cells were obtained from peripheral blood or lymph node biopsies at the time of diagnosis. Control samples were taken from healthy volunteer donors. The diagnoses were made according to French-American-British (FAB) criteria. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; AMML, acute myelomonocytic leukemia; AMoL, acute monocytic leukemia; n.a., not available; WBC, white blood cell count.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Activation Assay, Biomarker Discovery, Control

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Identification of Ser-193 as a novel proline-flanked phosphorylation site in human Stat5b. YT cells were stimulated without (−) or with (+) IL-2 for 15 min, and Stat5b proteins were immunoprecipitated (IP) from soluble cell lysates with α-Stat5b antibodies. Two sets of immunoprecipitations were separated by SDS-PAGE. One set was Coomassie Blue-stained (A), and the other was Western blotted (WB) (B) with α-phospho-Tyr (α-pY), α-phospho-Ser (pS726/731), or α-Stat5b antibodies. HC, heavy chain; LC, light chain. C, tandem mass spectra of a monophosphorylated peptide showing site localization of Ser-193, as indicated by asterisks. D, amino acid sequence alignment of the region surrounding Ser-193 (asterisk) from each human Stat protein using the ClustalW program (progressive alignment) (31). E, domain architecture of human Stat5 with known and newly identified (asterisk) serine and tyrosine phosphorylation sites. Numbers indicate amino acid residues of human Stat5 (a/b).

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Staining, Western Blot, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Phosphorylation of Stat5b Ser-193 displays rapid kinetics and is inducible by multiple cytokines. A, a phospho-specific polyclonal Stat5b Ser(P)-193 antibody was generated and tested by dot blot analysis using increasing amounts of Stat5b Ser-193 (CLAQLSPQERL), Ser(P)-193 (CLAQL(pS)PQERL), Ser-731 (KDQAPSPAVCP), and Ser(P)-731 (KDQAP(pS)PAVCP) peptides spotted onto PVDF membrane (where pS indicates phospho-serine). IB, immunoblotting. B, YT cells stimulated with IL-2 for 15 min were fluorescently labeled using α-phospho-Tyr (α-pY) Stat5 (Cy2, green), α-Ser(P)-193 (α-pS193) Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. For peptide competition analysis, the polyclonal Ser(P)-193 Stat5b antibody was preblocked with increasing amounts of non-phospho-peptide (lanes a–c) or phospho-peptide (lanes d–f) for 1 h at 4 °C followed by staining of YT cells treated with IL-2 for 15 min. C, YT cells were stimulated without (−) (lane a) or with IL-2 (+) from 0 to 60 min (lanes b–e) and fixed with cold methanol. Cells were then stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. Insets show a higher magnification view of α-Ser(P)-193 Stat5b (Cy3, red)-stained YT cells treated with IL-2 for the indicated time points. D, quiescent PHA-activated human PBMCs were stimulated with medium (−) (lane a) or IL-2 (lane b), IL-7 (lane c), IL-9 (lane d), or IL-15 (lane e) for 15 min and fixed with cold methanol. Confocal images using α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and overlay images are shown. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Phospho-proteomics, Generated, Dot Blot, Membrane, Western Blot, Labeling, Confocal Microscopy, Staining, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 undergoes phosphorylation in an mTOR-dependent manner. A, YT cells were left untreated (lanes a and b) or pretreated with increasing concentrations of rapamycin (1–100 nm) (lanes c–f) for 1 h followed by stimulation with IL-2 (lanes b–f) for 15 min. α-pY, α-phospho-Tyr; α-pS193, α-Ser(P)-193. B, YT cells were left untreated (lanes a and b) or pretreated with 10–200 nm PP242 hydrate for 1 h followed by stimulation with IL-2 for 15 min (lanes b–f) as indicated. The cells were fixed and stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Phospho-proteomics, Staining, Confocal Microscopy, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: PP2A, but not PP1, negatively regulates Stat5b Ser-193 phosphorylation in YT cells. A, cells were pretreated without (lanes a and b) or with DMSO (lanes c and d) or 50 nm CA (lanes e and f) for 1 h before stimulation without (lanes a, c, and e) or with IL-2 (lanes b, d, and f) for 15 min at 37 °C. The cells were fixed and stained with α-phospho-Tyr Stat5 (Cy2, green), α-Ser(P)-193 (α-pS193) Stat5b (Cy3, red), and DAPI (blue) and visualized by confocal microscopy. The overlay (bottom panel) shows co-localization of phospho-Tyr Stat5 and Ser(P)-193 Stat5b. NT, not treated. B, YT cells were left untreated (lanes a and b) or pretreated with 150 nm OA (lanes c and d), 25 nm FOS (lanes e and f), or 1 μm TAU (lanes g and h) for 1 h before stimulation without (−) or with (+) IL-2 for 15 min at 37 °C. The cells were fixed and analyzed using α-Ser(P)-193 Stat5b and DAPI as described above. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Phospho-proteomics, Staining, Confocal Microscopy, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 phosphorylation is required for its maximal DNA binding and transcriptional activity. A, HEK293 cells were transfected with IL-2Rβ, IL-2Rγ, Jak3, and Stat5b (WT, S193A, or S193E) and incubated for 48 h. The cells were then stimulated without (−) or with IL-2 (+) for 15 min. Stat5b WT (lanes a and b), Stat5b S193A (lanes c and d), and Stat5b S193E (lanes e and f) were blotted with α-phospho-Tyr (α-pY) Stat5 (upper panel) or total Stat5 (lower panel). WB, Western blot. B, nuclear extracts (5 μg) isolated from transfected HEK293 described in A were incubated with a 32P-radiolabeled oligonucleotide probe corresponding to the Stat5 binding site in the β-casein gene promoter. The extracts indicated were co-incubated with N-terminal directed α-Stat5 (lane h) or normal rabbit IgG (Cntrl) (lane i). The bracket indicates the location of free probe, and the arrows indicate the location of non-supershifted and supershifted Stat5b-DNA complexes. Representative data from two independent experiments are shown. C, HEK293 cells transfected as described in A were treated without (−) or with (+) IL-2 for 6 h. Control cells were transfected with Stat5b alone (lanes a and b). At 48 h after transfection, the cells were lysed, and luciferase activities were measured and normalized to β-galactosidase activity. Statistical significance was determined using analysis of variance (*, p < 0.05). Representative data from three independent experiments are shown. Error bars indicate S.D.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Transfection, Incubation, Western Blot, Isolation, Control, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Signal Transducer and Activator of Transcription 5b (Stat5b) Serine 193 Is a Novel Cytokine-induced Phospho-regulatory Site That Is Constitutively Activated in Primary Hematopoietic Malignancies *

doi: 10.1074/jbc.M111.319756

Figure Lengend Snippet: Stat5b Ser-193 is constitutively phosphorylated in HTLV-1-transformed tumor T-cell lines and primary leukemia and lymphoma patient tumor cells. A, immunofluorescent confocal microscopy was utilized to detect phospho-Tyr Stat5 (α-pY Stat5) and Ser(P)-193 Stat5b (α-pS193 Stat5b) in normal PHA-activated quiescent human PBMCs stimulated with IL-2 or without for 15 min (lanes a and b), non-PHA-activated PBMCs cells (lane c), MT-2 (lane d), HUT-102 (lane e), and HUT-78 (lane f). B, immunofluorescent confocal microscopy was utilized to detect phospho-Tyr Stat5 and Ser(P)-193 Stat5b in primary tumor cells isolated from patients diagnosed with ALL (lane d), AML (lane e), and B-cell lymphoma (lane f). Normal PHA-activated quiescent human PBMCs stimulated with IL-2 or without IL-2 for 15 min (lanes a and b) and non-PHA-activated PBMCs cells (lane c) served as controls. Immunofluorescent images were captured using PASCAL software on a Zeiss LSM 510 Meta confocal microscope at 63× magnification. Representative data from three independent experiments are shown.

Article Snippet: The anti-phospho-serine 193 Stat5b (α-Ser(P)-193 Stat5) rabbit polyclonal antibody was custom-generated and affinity-purified by GenScript (Piscataway, NJ) using the immunogen CLAQL(pS)PQERL (where pS indicates phospho-serine) coupled to keyhole limpet hemocyanin.

Techniques: Transformation Assay, Confocal Microscopy, Isolation, Software, Microscopy

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: List of primer sequences used for RT-PCR analysis.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques:

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: Increased activity of STAT3, which is required for T-cell suppression. (a 1 ) Neutrophils before and after induction were tested for STAT1, STAT3, STAT5, p-STAT1, p-STAT3, and p-STAT5 expression by WB. (a 2 ) Grayscale analysis of WB results by image J software; the result was expressed as the gray value of the target band/the gray value of the reference protein. Increased expression levels of STAT3 and p-STAT3 proteins were found in neutrophils AI compared with those before induction. (b) Changes of mRNA expression of STAT1, STAT3, and STAT5 in neutrophils before and after induction. The mRNA level of STAT3 in neutrophils AI was significantly higher than that in neutrophils before induction. (c 1 and c 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h; T-cell proliferation was evaluated by CFSE labeling, and unstimulated T cells were used as a negative control. The samples were analyzed by flow cytometry. Ruxolitinib addition almost completely abolished the ability of CD10 − neutrophils to suppress T-cell proliferation. (c 3 ) Representative flow cytometry data of T-cell proliferation from one individual in coculture and indirect coculture systems. The red area represented the proliferating T-cell fraction. (d 1 and d 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h, and apoptotic T cells were assessed by flow cytometry. CD3 + T cells cultured alone were used as a negative control. Ruxolitinib addition almost completely prevented T-cell apoptosis. (d 3 ) Representative flow cytometry data of CD3 + T-cell apoptosis experiment from one individual in the coculture or indirect coculture system. CD3 + T cells without PI or annexin V were used as a blank control. Apoptotic T cells were marked as CD3 + /annexin + /PI + . (e 1 ) WB analysis of the changes in STAT3 and p-STAT3 expression before and after ruxolitinib addition in the induced neutrophil population and (e 2 ) grayscale analysis of WB results by imageJ software. Ruxolitinib inhibited the expression of STAT3 and p-STAT3 proteins. (f) Extracellular production of Arg-1 protein was evaluated by ELISA at 48 h before and after the addition of ruxolitinib in neutrophils AI . Significantly decreased expression of extracellular Arg-1 in neutrophils AI treated with ruxolitinib. (g) Changes in intracellular Arg-1 fluorescence intensity were detected by flow cytometry at 48 h before and after ruxolitinib addition in neutrophils AI . Significantly decreased expression of intracellular Arg-1 in neutrophils AI treated with ruxolitinib, mainly in the CD10 − neutrophil subset. (h) Arg-1 activity was determined at 48 h before and after ruxolitinib addition in neutrophils AI . A significant decrease in Arg-1 activity was observed in the cells treated with ruxolitinib. (i) mRNA expression of Arg-1 was detected by RT-PCR in neutrophils AI at 48 h before and after ruxolitinib addition. A significant decrease was observed in the mRNA expression of Arg-1 in the cells treated with ruxolitinib. (j) mRNA expression of STAT3 was detected by RT-PCR in neutrophils at 48 h before and after ruxolitinib addition. The expression of STAT3 gene in CD10 − neutrophil subpopulation significantly decreased. Each experiment was repeated 3 times. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ✶ ✶ ✶ ✶ P < 0.0001, ns: P > 0.05. Ctrl: Neutrophils before induction, neutrophils AI : Neutrophils after induction, WB: Western blot, CFSE: Carboxyfluorescein succinimidyl ester, CD10 − neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 − cells, CD10 + neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 + cells.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques: Activity Assay, Expressing, Software, Labeling, Negative Control, Flow Cytometry, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot