Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-c , CD8 + T cells were treated with EPZ004777 for 48 hours. Western Blot showed H3K79me2 in CD8 + T cells ( a ). FACS demonstrated CD8 + T cell apoptosis ( b ) and cytokines ( c ). d , Western Blot showed H3K79me2 in Dot1l f/f and Dot1l −/− CD8 + T cells. e, f , Effect of DOT1L deletion on CD8 + T cells apoptosis ( e ) and cytokines ( f ). g-i , Effect of T cell DOT1L deficiency on MC38 growth ( g, h ) and T cell viability ( i ). j, k , Effect of methionine supplementation on Dot1l f/f and Dot1l −/− CD8 + T cells apoptosis ( j ) and cytokines ( k ). l , GSEA plot showed enriched apoptotic pathway in Dot1l −/− CD8 + T cells. m , Heat map showed Jak-Stats mRNA levels in mouse Dot1l −/− vs Dot1l f/f CD8 + T cells. n , Western blot showed STAT5 and p-STAT5 in Dot1l f/f ( f/f ) and Dot1l −/− (−/−) CD8 + T cells. o , Western blot showed STAT5 and p-STAT5 in CD8 + T cells cultured with fresh medium (FM), Supernatant (Sup), and Sup supplemented with different metabolites (Sup+). p , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells. q , ChIP assay showed H3K79me2 occupancy on the Stat5b promoter in CD8 + T cells cultured with FM, Sup, or Sup+Met. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Western Blot, Cell Culture

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a, b , H3K79me2 ( a ) and STAT5 ( b ) levels in CD8 + T cells from tumor draining lymph node (dLN) and tumor in B16F10 bearing mice. c, d , H3K79me2 ( c ) and STAT5 ( d ) levels in CD8 + T cells from spleen and tumor ascites in ID8 bearing mice. e , H3K79me2 levels in CD8 + T cells from healthy peripheral blood and human ovarian cancers ascites. f, g , H3K79me2 ( f ) and STAT5 ( g ) levels in CD8 + T cells from healthy human blood and human ovarian cancer omentum tissues. h, i , FACS showed H3K79me2 and STAT5 levels in human tumor infiltrating CD8 + T cells. j-m , Effect of methionine on human tumor infiltrating CD8 + T cells. Human colorectal cancer infiltrating CD8 + T cells were cultured with or without methionine. T cell cytokine production ( j, k ), H3K79me2 ( l ), and STAT5 ( m ) were analyzed by FACS. One representative of four is shown. n , Effect of methionine supplementation on apoptosis of tumor infiltrating CD8 + T cells and ID8 tumor cells in vivo. ID8 tumor bearing mice were treated with methionine or PBS. T cell and tumor cell apoptosis was determined by FACS. o , Methionine levels in ID8 tumor after methionine or PBS treatment. p-r: Effect of anti-PD-L1 on methionine-affected CT26 tumor progression. Mice bearing CT26 tumor were treated with anti-PD-L1, methionine, and their combination. Tumor volume ( p ), T cell tumor infiltration ( q ) and apoptosis ( r ) were assessed. Data are mean ± s.e.m. Information on sample sizes, experimental number, times, biological replicates, statistical tests, and P values is available in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Cell Culture, In Vivo

Journal: Nature

Article Title: Cancer SLC43A2 alters T cell methionine metabolism and histone methylation

doi: 10.1038/s41586-020-2682-1

Figure Lengend Snippet: a-e , Methionine supplementation restored T cell immunity in B16F10-bearing mice. Tumor growth ( a ), tumor infiltrating CD8 + T cell H3K79me2 ( b ) and STAT5 ( c ) were monitored. FACS showed intratumor CD8 + T cell apoptosis ( d ) and cytokines ( e ) . f-h , Methionine supplementation restored T cell immunity in ID8-bearing mice. Tumor growth was monitored by bioluminescence imaging ( f ). FACS showed ascites CD8 + T cell ( g ) and intratumor CD8 + T cell cytokines ( h ) . i-l , Studies on colorectal cancer patients treated with methionine. Western blot showed p-STAT5 and H3K79me2 in peripheral CD8 + T cells prior and post methionine treatment ( i ). FACS showed IL-2 + T cells ( j ), CD8 + T cell effector cytokines ( k ) and apoptosis ( l ) in patients prior and post methionine treatment. Data are mean ± s.e.m. Sample sizes (n), P values, statistical tests and number of times experiments were replicated are listed in ‘Statistics and reproducibility’.

Article Snippet: For STAT5, cells were stained with APC-anti-STAT5 (REA549, Miltenyi Biotec Inc., Bergisch Gladbach, Germany).

Techniques: Imaging, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: BMC Genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: Putative miRNAs targeting gene 3’UTRs. (A) Pairing of bta-miR-141 with the bovine STAT5 3’UTR sequence. (B) A target seed region of the miRNA binding site was found within the bovine HK2 3’UTR sequence.

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Sequencing, Binding Assay

Journal: BMC Genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: Expression of HK2 and STAT5 mRNA in the bovine mammary gland. Expression levels of HK2 and STAT5 in lactating and non-lactating bovine mammary glands were determined using real-time PCR. Gene fold changes were calculated using the 2 −ΔCt method, where ΔCt = (Ct HK2/STAT5 − Ct GAPDH ). n=3.

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Expression profiles of microRNAs from lactating and non-lactating bovine mammary glands and identification of miRNA related to lactation

doi: 10.1186/1471-2164-13-731

Figure Lengend Snippet: STAT5 protein is regulated by miR-141. STAT5 and β-actin protein expression was determined by western blotting. Mac-T cells were transfected with an miR-141 inhibitor, an miR-141 mimic, an inhibitor negative control (INC) and a negative control (NC). Cell lysates were collected after 24 hours and analyzed by western blotting. Beta-actin served as the internal control. The protein fragment intensity was quantified using Imagpro-Plus software, and IOD was used to represent the protein level. Error bars represent SD. n=3. a-c means different superscripts differ ( P <0.05).

Article Snippet: Antibodies for STAT5 and β-actin were manufactured by Boster (China), and the HK2 antibody was purchased from Santa Cruz (USA).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Control, Software

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