Antibody data
- Antibody Data
- Antigen structure
- References [9]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [6]
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- Product number
- 11-9010-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-STAT5 (Tyr694) Monoclonal Antibody (SRBCZX), FITC, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: This SRBCZX monoclonal antibody recognizes signal transducer and activator of transcription 5 (STAT5) when phosphorylated on tyrosine 694. STAT proteins are activated by ligand binding to receptors, such as cytokine receptors, that associate with Janus kinase (JAK) family members. Following their phosphorylation by JAKs, STAT proteins translocate to the nucleus where they bind to DNA and regulate transcription of specific genes in a cell type- and cytokine-specific manner. In response to cytokines that signal through the common gamma chain such as IL-2, IL-7, and IL-15, STAT5 is phosphorylated on tyrosine 694 by JAK1 and JAK3. Cytokines such as IL-3, IL-5, and GM-CSF that signal via the common beta chain induce STAT5 phosphorylation on tyrosine 694 by JAK 2. Phosphorylation of STAT5 on tyrosine 694 is essential for STAT5 dimer formation, nuclear translocation, and DNA binding activity. Specificity of this SRBCZX clone was determined by ELISA and flow cytometry. Applications Reported: This SRBCZX antibody has been reported for use in intracellular staining followed by flow cytometric analysis. Applications Tested: This SRBCZX antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of stimulated U937 cells. This can be used at 5 µL (1 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. Staining Protocol: We recommend using Protocol C: Two-step protocol: Fixation/Methanol. Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins and Protocol B: One-step protocol: intracellular (nuclear) proteins cannot be used. All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the Best Protocols Section under the Resources tab online. Excitation: 488 nm; Emission: 520 nm; Laser: Blue Laser. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human, Mouse
- Host
- Mouse
- Conjugate
- Green dye
- Isotype
- IgG
- Antibody clone number
- SRBCZX
- Vial size
- 100 Tests
- Concentration
- 5 µL/Test
- Storage
- 4° C, store in dark, DO NOT FREEZE!
Submitted references GM-CSF impairs erythropoiesis by disrupting erythroblastic island formation via macrophages.
Oleic acid restores suppressive defects in tissue-resident FOXP3 Tregs from patients with multiple sclerosis.
Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation.
Inactivation of mTORC1 Signaling in Osterix-Expressing Cells Impairs B-cell Differentiation.
Erythropoietin enhances Kupffer cell number and activity in the challenged liver.
PTEN drives Th17 cell differentiation by preventing IL-2 production.
Expression and function of hematopoiesis-stimulating factor receptors on the GPI(-) and GPI(+) hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome.
Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells.
Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency.
Cao W, Fan W, Wang F, Zhang Y, Wu G, Shi X, Shi JX, Gao F, Yan M, Guo R, Li Y, Li W, Du C, Jiang Z
Journal of translational medicine 2022 Jan 3;20(1):11
Journal of translational medicine 2022 Jan 3;20(1):11
Oleic acid restores suppressive defects in tissue-resident FOXP3 Tregs from patients with multiple sclerosis.
Pompura SL, Wagner A, Kitz A, LaPerche J, Yosef N, Dominguez-Villar M, Hafler DA
The Journal of clinical investigation 2021 Jan 19;131(2)
The Journal of clinical investigation 2021 Jan 19;131(2)
Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation.
Ferrao RD, Wallweber HJ, Lupardus PJ
eLife 2018 Jul 25;7
eLife 2018 Jul 25;7
Inactivation of mTORC1 Signaling in Osterix-Expressing Cells Impairs B-cell Differentiation.
Wang Y, Xiao M, Tao C, Chen J, Wang Z, Yang J, Chen Z, Zou Z, Liu A, Cai D, Jiang Y, Ding C, Li M, Bai X
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2018 Apr;33(4):732-742
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2018 Apr;33(4):732-742
Erythropoietin enhances Kupffer cell number and activity in the challenged liver.
Gilboa D, Haim-Ohana Y, Deshet-Unger N, Ben-Califa N, Hiram-Bab S, Reuveni D, Zigmond E, Gassmann M, Gabet Y, Varol C, Neumann D
Scientific reports 2017 Sep 4;7(1):10379
Scientific reports 2017 Sep 4;7(1):10379
PTEN drives Th17 cell differentiation by preventing IL-2 production.
Kim HS, Jang SW, Lee W, Kim K, Sohn H, Hwang SS, Lee GR
The Journal of experimental medicine 2017 Nov 6;214(11):3381-3398
The Journal of experimental medicine 2017 Nov 6;214(11):3381-3398
Expression and function of hematopoiesis-stimulating factor receptors on the GPI(-) and GPI(+) hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome.
Fu R, Ding SX, Liu YI, Li LJ, Liu H, Wang HL, Zhang T, Shao ZH
Experimental and therapeutic medicine 2016 May;11(5):1668-1672
Experimental and therapeutic medicine 2016 May;11(5):1668-1672
Monomethylarsonous acid (MMA+3) Inhibits IL-7 Signaling in Mouse Pre-B Cells.
Ezeh PC, Xu H, Lauer FT, Liu KJ, Hudson LG, Burchiel SW
Toxicological sciences : an official journal of the Society of Toxicology 2016 Feb;149(2):289-99
Toxicological sciences : an official journal of the Society of Toxicology 2016 Feb;149(2):289-99
Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency.
Mucci A, Kunkiel J, Suzuki T, Brennig S, Glage S, Kühnel MP, Ackermann M, Happle C, Kuhn A, Schambach A, Trapnell BC, Hansen G, Moritz T, Lachmann N
Stem cell reports 2016 Aug 9;7(2):292-305
Stem cell reports 2016 Aug 9;7(2):292-305
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Intracellular staining of untreated (orange histogram) or 15-minute Human GM-CSF Recombinant Protein-treated (purple histogram, Product # 14-8339-62) U937 cells with Anti-Human/Mouse phospho-STAT5 (Y694) FITC. Total viable cells were used for analysis.
- Conjugate
- Green dye
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- Conjugate
- Green dye
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 1 EPO regulates EPO-R expression and elicits downstream signalling in RKC-2 cells. All graphs represent mean +- SEM. ( A-B) RKC-2 cells were cultured in the presence or absence of 5 U/ml EPO for 24 h. ( A ) EPO-R transcript levels were evaluated by RT-PCR, N = 5-8, *p < 0.05. Rat BMDM (positive control) were considered as 1. ( B ) EPO-R surface expression was evaluated by flow cytometry. Top: Grey and black line histograms depict surface EPO-R in control (considered as 1) and EPO-treated cells, respectively. Full histogram depicts FITC conjugated Goat anti mouse antibody. Bottom: Quantification of surface EPO-R, N = 7, **p < 0.01. ( C-D ) RKC-2 cells were stimulated for 0, 10 and 30 min with 10 U/ml EPO. Top: Grey, black and dashed histograms represent 0, 10 and 30 min EPO-treated cells, respectively. Bottom: The graphs depict quantification of mean fluorescence intensity (MFI) of flow cytometry analysis for p-ERK1/2 and p-STAT5, ( C ) and ( D ), respectively. Levels at 0 min were considered as 1, N = 4-5, **p < 0.01, *p < 0.05.
- Conjugate
- Green dye
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 5. EPOR and LEPR switch residues are required for Epo and Leptin-induced STAT phosphorylation. ( a,b ) Stable Ba/F3 cell lines expressing wild-type, full length mouse EPOR or LEPR were generated and analyzed for STAT phosphorylation by flow cytometry after stimulation with EPO or leptin. ( a ) Representative plots comparing phospho-STAT5 staining of parental Ba/F3 cells or Ba/F3 cells expressing EPOR. Cells were stimulated with 1 nM mouse EPO for 15 min before fixation, staining, and analysis. ( b ) Representative plots comparing phospho-STAT3 staining of parental Ba/F3 cells or Ba/F3 cells expressing LEPR. Cells were stimulated with 100 pM mouse Leptin for 4 hr prior to fixation, staining, and analysis. ( c ) Analysis of STAT5 phosphorylation by flow cytometry for EPOR wild-type, switch region, and box1 mutants, stimulated with 1 nM EPO as in ( a ). Mean levels of STAT5 phosphorylation were assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. ( d ) Analysis of STAT3 phosphorylation by flow cytometry for LEPR wild-type, switch region, and box1 mutants, stimulated with 100 pM Leptin, as in ( b ). Mean levels of STAT3 phosphorylation was assessed in three separate experiments, with wild-type signal representing 100% in all three experiments. Error bars represent standard error of the mean (SEM). Figure 5--figure supplement 1. Analysis of Ba/F3 stable cell lines expressing EPOR or LEPR mutants. ( a ) Alignment of human
- Conjugate
- Green dye
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 5--figure supplement 1. Analysis of Ba/F3 stable cell lines expressing EPOR or LEPR mutants. ( a ) Alignment of human and mouse sequences for EPOR and LEPR. Note the numbering differences for mouse versus human (minus one residue for EPOR and minus two residues for LEPR). Residues in the switch region and box1, which are mutated in our experiments, are completely conserved between human and mouse. ( b-c ) Receptor surface staining for stable cell lines expressing ( b ) EPOR variants and ( c ) LEPR variants. Plots represent cumulative events (Y axis) at or below a given staining level (X axis). ( d ) Representative raw flow cytometry histograms showing the phospho-STAT5 (EPOR) or phospho-STAT3 (LEPR) staining in unstimulated cells (black trace) or cells stimulated with cytokine (red trace) as described in Materials and methods.
- Conjugate
- Green dye
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 3. The P-STAT5 MFI in the (A) GPI + cells and GPI - cells of PNH/AA patients and (B) GPI + cells of normal controls with or without stimulation by G-CSF or SCF. (C) The MFI of P-STAT5 in GPI + and GPI - cells of the bone marrow before and after G-CSF or SCF stimulation. G-CSF, granulocyte colony-stimulating factor; SCF, stem cell factor; P-STAT5, phosphorylated signal transducer and activator of transcription 5; MFI, mean fluorescence intensity; GPI, glycophosphatidylinositol; PNH, paroxysmal nocturnal hemoglobinuria; CD, cluster of differentiation.
- Conjugate
- Green dye
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 6 Disease Modeling of herPAP Using miPAP iPSCs (A) Number of CD41 + progenitor-derived colonies in methylcellulose-based clonogenic assays employing complete (IL-6, erythropoietin, SCF, IL-3, and supplemented with 20 ng/ml human G-CSF + 20 ng/ml murine GM-CSF) or basic medium (50 ng/ml murine GM-CSF only; independent experiments, n = 3 miPAP1 and 2, n = 2 CD45.1 iPSC, mean +- SD) and representative pictures of colonies. Scale bars, 500 mum. (B) GM-CSF clearance assay comparing miPAP-Mphi with CD45.1(10.3) iPSC-Mphi, BMlin - -Mphi, and no cells incubated with 2 ng/ml GM-CSF: at indicated time points (0, 4, 10, 24, and 30 hr) GM-CSF concentrations in supernatants were analyzed by ELISA, normalized to 0 hr (independent experiments, n = 3 BMlin - and CD45.1 iPSC, n = 2 miPAP1 and 2, mean +- SD). (C and D) Flow cytometry plots of STAT5 phosphorylation levels upon stimulation with mGM-CSF (C) and (D) summary of mean fluorescence intensity (MFI) data (independent experiments, n = 3 BMlin - and CD45.1 iPSC, n = 2 miPAP1 and 2, mean +- SD). ns, not significant; ** p
- Conjugate
- Green dye