Antibody data
- Antibody Data
- Antigen structure
- References [5]
- Comments [0]
- Validations
- Flow cytometry [1]
- Other assay [6]
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- Product number
- 61-9010-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-STAT5 (Tyr694) Monoclonal Antibody (SRBCZX), PE-eFluor™ 610, 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 normal human peripheral blood cells. This can be used at 5 µL (0.5 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. PE-eFluor® 610 can be excited with laser lines from 488-561 nm and emits at 607 nm. We recommend using a 610/20 band pass filter (equivalent to PE-Texas Red®). Please make sure that your instrument is capable of detecting this fluorochome. Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light. Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically. Excitation: 488-561 nm; Emission: 607 nm; Laser: Blue Laser, Green Laser, Yellow-Green Laser. Filtration: 0.2 µm post-manufacturing filtered.
- Reactivity
- Human, Mouse
- Host
- Mouse
- 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 Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation.
Erythropoietin enhances Kupffer cell number and activity in the challenged liver.
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.
Ferrao RD, Wallweber HJ, Lupardus PJ
eLife 2018 Jul 25;7
eLife 2018 Jul 25;7
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
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
- TOP: Intracellular staining of untreated (left) or 15-minute IL-15-treated (right) normal human peripheral blood cells with Anti-Canine/Human CD94 APC (Product # 17-5094-42) and Anti-Human/Mouse phospho-STAT5 (Y694) PE-eFluor® 610. Cells in the lymphocyte gate were used for analysis. BOTTOM: Intracellular staining of untreated (orange histogram) or 15-minute IL-15-treated (purple histogram) normal human peripheral blood cells with Anti-Human/Mouse phospho-STAT5 (Y694) PE-eFluor® 610. CD94+ cells in the lymphocyte gate were used for analysis. In both panels, cells were stained using the Fixation/Methanol protocol.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- NULL
- 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.
- 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
- 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.
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- Invitrogen Antibodies (provider)
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- 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.
- 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