Phospho-STAT5A (Y694) Recombinant Monoclonal Antibody

What is the specificity of Phospho-STAT5A/B (Y694/Y699) antibodies?

Phospho-STAT5A/B (Y694/Y699) antibodies specifically detect STAT5A/B when phosphorylated at tyrosine 694 (STAT5A) or tyrosine 699 (STAT5B). The antibodies are validated through direct ELISAs and Western blots . These antibodies typically recognize the phosphopeptide containing the human STAT5B Y699 site, which is identical in amino acid sequence to the corresponding region of human STAT5A containing Y694 . This high specificity ensures accurate detection of activated STAT5 in experimental systems.

What cellular models are suitable for studying Phospho-STAT5A/B signaling?

Several well-validated cellular models are appropriate for studying Phospho-STAT5A/B signaling:

• Daudi human Burkitt's lymphoma cell line: Shows robust STAT5 phosphorylation in response to IFN-alpha stimulation (500 U/mL for 20 minutes), making it an excellent model for studying cytokine-induced STAT5 activation .

• HeLa human cervical epithelial carcinoma cell line: Demonstrates STAT5 phosphorylation upon treatment with IFN-alpha or human epidermal growth factor (hEGF) .

• TF-1 human erythroleukemic cell line: Requires GM-CSF supplementation to maintain persistent STAT5 phosphorylation, making it useful for studying STAT5 activation dynamics .

• MCF-7 and MDA-MB-231 breast cancer cell lines: Useful for breast cancer-specific STAT5 signaling research .

• Acute myeloid leukemia (AML) cell lines: Different AML cell lines show varying levels of STAT5 phosphorylation, allowing for comparative studies of hematological malignancies .

What are the validated detection methods for Phospho-STAT5A/B?

Multiple detection methods have been validated for Phospho-STAT5A/B:

• Western Blot: Detects a specific band at approximately 95 kDa in stimulated cells. Recommended antibody concentration is 0.1-2 μg/mL, depending on the specific antibody clone .

• Flow Cytometry: Effective for quantitative analysis of phosphorylated STAT5 at the single-cell level. Requires cell fixation with paraformaldehyde and permeabilization with methanol .

• Immunocytochemistry: Useful for visualizing subcellular localization of phosphorylated STAT5, particularly nuclear translocation following activation. Recommended antibody concentration is 0.3-25 μg/mL .

• CyTOF (Mass Cytometry): Some antibody clones are CyTOF-ready, enabling multi-parameter analysis of signaling pathways .

How should samples be prepared for optimal Phospho-STAT5A/B detection?

For optimal detection of Phospho-STAT5A/B, sample preparation is crucial:

For Western Blot analysis:

• Stimulate cells with appropriate cytokines (e.g., 500 U/mL IFN-alpha for 20 minutes for Daudi or HeLa cells) .

• Lyse cells under reducing conditions using appropriate buffer systems (e.g., Immunoblot Buffer Group 1) .

• Run samples on SDS-PAGE and transfer to PVDF membrane.

• Probe with 0.1-2 μg/mL of anti-Phospho-STAT5A/B antibody .

• Use appropriate HRP-conjugated secondary antibody for detection.

For Flow Cytometry:

• Apply appropriate stimulation to cells.

• Fix cells with paraformaldehyde.

• Permeabilize with methanol to facilitate intracellular staining.

• Stain with primary antibody followed by fluorophore-conjugated secondary antibody (e.g., APC-conjugated Anti-Rabbit IgG) .

For Immunocytochemistry:

• Fix cells by immersion fixation.

• Permeabilize cell membranes.

• Apply primary antibody at 0.3-25 μg/mL.

• Counterstain with DAPI for nuclear visualization .

What are the optimal storage conditions for Phospho-STAT5A/B antibodies?

To maintain antibody functionality, follow these storage guidelines:

• As supplied: Store at -20 to -70°C for up to 12 months from the date of receipt .

• After reconstitution:

  • Short-term storage (up to 1 month): 2 to 8°C under sterile conditions

  • Long-term storage (up to 6 months): -20 to -70°C under sterile conditions

Important considerations:

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce sensitivity .

• For lyophilized antibodies, reconstitute at 0.5 mg/mL in sterile PBS. For liquid formulations, refer to the Certificate of Analysis for the specific concentration .

• Aliquot reconstituted antibody to minimize freeze-thaw cycles.

What cytokines or growth factors effectively induce STAT5 phosphorylation?

Several cytokines and growth factors have been validated for inducing STAT5 phosphorylation:

Additional cytokines known to activate STAT5 signaling include IL-22 and IL-6, though specific experimental conditions were not detailed in the search results .

How does STAT5 phosphorylation regulate the expression of other proteins?

STAT5 phosphorylation at Y694/Y699 is involved in complex regulatory relationships with other proteins:

• Regulation of NPM1 (Nucleophosmin): Phosphorylated STAT5 negatively regulates NPM1 expression. Induction of STAT5 phosphorylation (via IL-3 stimulation) decreases NPM1 levels, while inhibition of STAT5 phosphorylation enhances NPM1 expression . This relationship has been demonstrated in multiple cell lines including TF-1, HeLa, and HEK 293T cells.

• p53 Regulation: Phosphorylated STAT5 regulates p53 expression via BRCA1 and NPM1. This STAT5 signaling pathway reveals potential therapeutic targets for anticancer treatment .

• Reciprocal Regulatory Relationship: There exists a mutually regulatory relationship between STAT5 and NPM1. While phosphorylated STAT5 diminishes NPM1 expression, NPM1 negatively regulates STAT5 phosphorylation while preserving unphosphorylated STAT5 levels .

These regulatory relationships highlight the complexity of STAT5 signaling networks and their importance in various cellular processes including cell survival, proliferation, and cancer development.

What are the key differences between detecting phosphorylated STAT5A versus total STAT5A?

Detecting phosphorylated STAT5A versus total STAT5A requires different experimental considerations:

Phosphorylated STAT5A Detection:

• Requires phospho-specific antibodies that recognize the Y694 phosphorylation site .

• Sample timing is critical - samples must be collected promptly after stimulation as phosphorylation is transient.

• Phosphatase inhibitors must be included in lysis buffers to preserve phosphorylation status.

• Phosphorylated STAT5A is predominantly localized to the nucleus after activation, requiring nuclear extraction or whole-cell lysis protocols .

• Stimulation with specific cytokines or growth factors is often necessary to induce detectable phosphorylation .

Total STAT5A Detection:

• Uses antibodies that recognize STAT5A regardless of phosphorylation status.

• Sample timing is less critical as total protein levels are generally more stable.

• Shows both cytoplasmic and nuclear localization, with distribution patterns changing upon activation.

• Can be detected without prior stimulation, though comparative studies often examine both basal and stimulated conditions.

Understanding these differences is crucial for experimental design and interpretation, particularly in studies examining STAT5 activation dynamics or comparing activation states across different conditions or cell types.

How can STAT5 phosphorylation be specifically inhibited in experimental systems?

Specific inhibition of STAT5 phosphorylation can be achieved through several approaches:

• Small Molecule Inhibitors: Three validated STAT5 inhibitors have been shown to specifically block STAT5 phosphorylation at Y694:

  • Compound 573108

  • AC-3-19

  • AC-4-130

  These inhibitors decrease P-STAT5 levels and consequently increase NPM1 expression . They can also reverse cytokine-induced (e.g., IL-3) STAT5 phosphorylation.

• Genetic Approaches:

  • STAT5-targeting shRNA: Lentiviral vectors carrying STAT5-specific shRNA effectively reduce STAT5 phosphorylation levels .

  • STAT5A Y694F mutant expression: This phosphorylation-deficient mutant can act as a dominant negative by competing with endogenous STAT5, preventing phosphorylation-dependent effects .

• Cytokine Receptor Antagonists: Blocking the receptors that activate STAT5 (e.g., IL-3 receptor, GM-CSF receptor) can indirectly inhibit STAT5 phosphorylation.

These approaches provide researchers with complementary tools to dissect STAT5 signaling pathways, offering flexibility in experimental design and interpretation.

What are common causes of false negative results when detecting Phospho-STAT5A/B?

False negative results when detecting Phospho-STAT5A/B can occur for several reasons:

• Rapid Dephosphorylation: STAT5 phosphorylation is dynamic and can be rapidly reversed by cellular phosphatases. Ensure samples are processed quickly and include phosphatase inhibitors in lysis buffers.

• Inadequate Stimulation: Insufficient concentration or duration of cytokine/growth factor stimulation. For example, IFN-alpha should be used at 500 U/mL for at least 20 minutes for optimal STAT5 phosphorylation in Daudi cells .

• Improper Sample Preparation: For intracellular detection by flow cytometry, inadequate fixation (paraformaldehyde) or permeabilization (methanol) can prevent antibody access to the phosphorylated epitope .

• Antibody Degradation: Repeated freeze-thaw cycles or improper storage conditions can reduce antibody sensitivity .

• Cell Type-Specific Activation Requirements: Different cell lines may require different stimuli or concentrations. For instance, TF-1 cells require GM-CSF for persistent STAT5 phosphorylation, while HeLa cells respond to both IFN-alpha and hEGF .

• Competing Signaling Pathways: Activation of pathways that negatively regulate STAT5 phosphorylation, such as SOCS proteins or certain phosphatases, can suppress the signal.

How can researchers distinguish between STAT5A and STAT5B phosphorylation?

• Isoform-Specific Knockdown: Use siRNA or shRNA specifically targeting either STAT5A or STAT5B, followed by detection with a pan-phospho-STAT5A/B antibody. The reduction in signal intensity can indicate the relative contribution of each isoform.

• Isoform-Specific Expression: Ectopically express tagged versions (e.g., RFP-tagged wild-type STAT5A) in cellular models . This approach allows tracking of the specific isoform's phosphorylation dynamics.

• Mass Spectrometry: For definitive identification, phosphopeptide analysis by mass spectrometry can distinguish between STAT5A and STAT5B based on differences in the surrounding amino acid sequences.

• Cell Type Considerations: Some cell types preferentially express one isoform over the other. Knowledge of the dominant isoform in your experimental system can help interpretation.

• Sequential Immunoprecipitation: First immunoprecipitate with an isoform-specific antibody, then detect phosphorylation status with the phospho-specific antibody.

It's important to note that most phenotypic effects and experimental findings relate to both isoforms due to their functional redundancy in many contexts.

What controls should be included when studying STAT5 phosphorylation?

Rigorous experimental design for studying STAT5 phosphorylation should include these essential controls:

• Positive Controls:

  • Stimulated samples: Cells treated with known STAT5 activators (e.g., IFN-alpha for Daudi and HeLa cells, IL-3 for TF-1 cells)

  • Cell lines with constitutive STAT5 activation (e.g., certain AML cell lines)

• Negative Controls:

  • Unstimulated samples from the same cell line

  • Samples treated with specific STAT5 inhibitors (573108, AC-3-19, or AC-4-130)

  • Samples expressing STAT5A Y694F phosphorylation-deficient mutant

• Specificity Controls:

  • Peptide competition assay: Pre-incubation of antibody with phosphopeptide should abolish signal

  • Phosphatase treatment: Sample treatment with λ-phosphatase should eliminate phospho-specific signal

• Loading/Processing Controls:

  • Total STAT5 detection on the same samples to normalize phosphorylation levels

  • Housekeeping proteins (e.g., β-actin, GAPDH) for Western blot loading control

  • For flow cytometry, isotype control antibodies to establish background staining levels

• Biological Replicates:

  • Independent repeats to ensure reproducibility and enable statistical analysis

How does STAT5 phosphorylation contribute to cancer development and progression?

STAT5 phosphorylation plays significant roles in cancer development and progression through multiple mechanisms:

• Transcriptional Regulation: Phosphorylated STAT5 translocates to the nucleus and activates transcription of specific genes involved in cell survival, proliferation, angiogenesis, and metastasis in both hematopoietic and non-hematopoietic cancers .

• Prognostic Significance: STAT5 phosphorylation status can serve as a prognostic marker in certain cancers, including breast cancer .

• Oncogenic Transformation: Overexpression of STAT5 promotes breast cancer formation in mice, highlighting its causal role in carcinogenesis .

• Interaction with Tumor Suppressors: Phosphorylated STAT5 regulates p53 expression through a pathway involving BRCA1, potentially influencing cellular responses to DNA damage and apoptotic signals .

• NPM1 Regulation: The reciprocal regulatory relationship between phosphorylated STAT5 and NPM1 forms a complex network with apoptosis-related proteins, including p53, MDM2, and Arf, affecting cancer cell survival and treatment response .

• Hematological Malignancies: Different AML cell lines show varying levels of STAT5 phosphorylation, suggesting heterogeneity in STAT5 activation that may contribute to disease progression and treatment response .

These findings highlight STAT5 phosphorylation as a potential therapeutic target, with STAT5 inhibitors showing promise in preclinical cancer models.

What are the technical advances in multiplexed detection of STAT5 with other signaling proteins?

Recent technical advances have enhanced researchers' ability to simultaneously analyze STAT5 and other signaling proteins:

• CyTOF (Mass Cytometry): Some Phospho-STAT5A/B antibodies are now CyTOF-ready, enabling multi-parameter analysis of signaling pathways at single-cell resolution . This technology allows simultaneous detection of dozens of parameters without fluorescence spillover concerns.

• Multiplexed Flow Cytometry: Advanced flow cytometry protocols now enable concurrent detection of multiple phosphorylated proteins, including STAT5, STAT3, and receptor tyrosine kinases, providing comprehensive signaling profiles.

• Co-Immunoprecipitation Studies: Research has demonstrated physical interactions between phosphorylated STAT5 and other proteins such as NPM1, enabling investigation of signaling complexes .

• Multiplexed Western Blotting: Techniques like Jess™ or Wes™ automated Western blotting systems allow quantitative analysis of multiple proteins from minimal sample amounts, facilitating comparative studies of signaling pathways.

• Phospho-Proteomics: Mass spectrometry-based phospho-proteomics approaches can identify hundreds to thousands of phosphorylation events simultaneously, placing STAT5 phosphorylation in broader signaling contexts.

These technical advances provide researchers with powerful tools to investigate STAT5 signaling in complex biological systems, revealing new insights into pathway cross-talk and integration.

How can phospho-STAT5A/B antibodies be employed in preclinical drug development?

Phospho-STAT5A/B antibodies serve as valuable tools in preclinical drug development through several applications:

• Target Validation: Confirming STAT5's role in disease models by correlating phosphorylation status with disease progression or phenotypic outcomes.

• Compound Screening: High-throughput screening of potential STAT5 inhibitors using phospho-STAT5 levels as a readout. The three STAT5 inhibitors mentioned (573108, AC-3-19, and AC-4-130) were likely developed using such approaches .

• Mechanism of Action Studies: Determining whether novel therapeutics directly or indirectly affect STAT5 signaling, providing insight into their molecular mechanisms.

• Pharmacodynamic Biomarkers: Using phospho-STAT5 levels to monitor drug efficacy in preclinical models and potentially translating this to clinical studies.

• Resistance Mechanisms: Investigating whether STAT5 activation contributes to resistance against targeted therapies or conventional treatments.

• Combination Therapy Rationale: Identifying synergistic drug combinations by studying how different compounds affect STAT5 phosphorylation in combination with other pathway modulators.

• Patient Stratification Strategies: Developing assays to identify patient populations likely to respond to STAT5-targeted therapies based on baseline phosphorylation status.

The discovery that phosphorylated STAT5 regulates p53 expression via NPM1 opens new therapeutic avenues for anticancer treatment, highlighting the value of phospho-STAT5A/B antibodies in identifying novel drug targets and understanding complex signaling networks .