STAT5A Antibody

What is STAT5A and why is it important in research?

STAT5A is one of two closely related genes (alongside STAT5B) that belong to the STAT family of transcription factors. STAT5A mediates key physiological effects of prolactin and growth hormone in mammary glands and plays crucial roles in hematopoiesis, immune cell function, and cancer biology . STAT5A becomes activated through tyrosine phosphorylation, which enables its dimerization and nuclear translocation to regulate target gene expression .

The importance of STAT5A in research stems from its involvement in:

• Regulation of T cell activation and proliferation

• Hematopoietic stem cell self-renewal

• Breast cancer development and progression

• Erythroid differentiation and erythropoiesis

How do STAT5A antibodies differ from STAT5B-specific antibodies?

STAT5A and STAT5B share over 90% sequence identity, differing primarily in their carboxyl terminus . When selecting antibodies for research:

Researchers should select antibodies raised against unique epitopes when paralog specificity is required. For example, studies by Villarino et al. utilized specific antibodies to distinguish the genomic distribution and functional differences between STAT5A and STAT5B in immune cells .

What are the recommended protocols for detecting STAT5A by Western blotting?

For optimal Western blot detection of STAT5A:

• Prepare cell lysates in the presence of protease and phosphatase inhibitors

• Use 7-9% SDS-PAGE gels and run for extended periods (4-5 hours) for better separation

• Transfer proteins to nitrocellulose or PVDF membranes

• Block with 5% BSA or non-fat milk

• Use STAT5A-specific antibodies at recommended dilutions (typically 1:1000-1:8000)

• Detect with appropriate secondary antibodies and visualization methods

For phosphorylated STAT5A detection, stimulate cells with appropriate cytokines (IL-2, IL-3, prolactin, etc.) before lysis and use phospho-specific antibodies that recognize phosphorylated tyrosine 694 (pY694) .

The observed molecular weight for STAT5A is approximately 90-95 kDa, though variants such as the Δ5 STAT5A (missing exon 5) will migrate at a slightly lower molecular weight .

How can I distinguish between STAT5A homodimers, STAT5B homodimers, and STAT5A:STAT5B heterodimers?

Distinguishing between the different STAT5 dimer configurations requires specialized techniques:

Electrophoretic Mobility Shift Assay (EMSA) with Supershift:

• Prepare nuclear extracts from cells of interest

• Perform EMSA using labeled probes containing STAT5-binding sites

• Add specific antibodies against STAT5A, STAT5B, or both

• Analyze the resulting supershifts:

  • Anti-STAT5A antibodies will supershift STAT5A homodimers and STAT5A:STAT5B heterodimers

  • Anti-STAT5B antibodies will supershift STAT5B homodimers and STAT5A:STAT5B heterodimers

  • Combined antibodies will supershift all complexes

Sequential Immunoprecipitation:

• Perform first immunoprecipitation with anti-STAT5A antibody

• Analyze a portion of the immunoprecipitate by Western blotting with both anti-STAT5A and anti-STAT5B antibodies

• Perform a second immunoprecipitation on the supernatant with anti-STAT5B antibody

• Analyze by Western blotting to identify remaining STAT5B homodimers

Research by Liu et al. demonstrated that distinct complexes are observed in the anti-STAT5A supershifted material that are not present in anti-STAT5B supershifts, indicating structural differences between homo- and heterodimers .

What approaches can be used to study STAT5A localization in different subcellular compartments?

STAT5A exhibits dynamic subcellular localization, with important functions in the nucleus, cytoplasm, and mitochondria. Different approaches can reveal this compartmentalization:

Subcellular Fractionation:

• Separate nuclear, cytoplasmic, and mitochondrial fractions using density-gradient centrifugation

• Verify fraction purity using compartment-specific markers (e.g., TATA binding protein for nucleus, COX4 for mitochondria)

• Analyze STAT5A content in each fraction by Western blotting

• Quantify relative distribution (e.g., research shows ~0.69% of STAT5A localizes to mitochondria, 96.72% to cytoplasm, and 2.59% to nucleus in certain cell types)

Protease Protection Assay:
To confirm internal mitochondrial localization (versus outer membrane association):

• Isolate mitochondrial fractions

• Treat with proteinase K with or without Triton X-100

• Analyze by Western blotting (proteins inside mitochondria will be protected from proteinase K unless Triton X-100 is added)

Immunofluorescence Microscopy:

• Fix cells with paraformaldehyde or methanol

• Perform immunostaining with STAT5A-specific antibodies

• Use compartment-specific markers for colocalization studies

• Analyze using confocal microscopy

Artificial Targeting:
Construct fusion proteins with compartment-specific targeting sequences (e.g., mitochondrial targeting sequence from NDUFV2) to study functions in specific locations .

How can I accurately quantify STAT5A protein expression levels in tissue samples?

Multiple approaches can be used for accurate quantification of STAT5A protein levels in tissues:

AQUA (Automated Quantitative Analysis):

• Perform immunofluorescent staining on formalin-fixed, paraffin-embedded tissues

• Use STAT5A-specific antibodies (typical dilution 1:8,000)

• Apply the AQUA/PM2000 platform for automated quantification

• This approach provides objective, continuous scoring rather than categorical assessment

DAB-Chromogen Immunohistochemistry with Digital Analysis:

• Perform antigen retrieval using citric acid buffer (pH 6.0)

• Incubate with STAT5A antibody (1:8,000 dilution) for 30 minutes

• Develop with DAB substrate

• Quantify using digital pathology platforms

• Distinguish between nuclear (transcriptionally active) and cytoplasmic STAT5A

Western Blotting with Internal Controls:

• Extract proteins from tissue samples

• Load equal amounts of protein (verified by total protein staining)

• Include recombinant STAT5A standards for calibration

• Use housekeeping proteins as loading controls

• Perform densitometry for quantification

Research by Peck et al. demonstrated that lower levels of nuclear STAT5A protein in breast cancer are associated with worse clinical outcomes, highlighting the importance of accurate quantification and subcellular localization assessment .

How do I interpret contradictory results between STAT5A phosphorylation and downstream functional outcomes?

Researchers may encounter situations where STAT5A phosphorylation status doesn't correlate with expected functional outcomes. Several factors may explain these discrepancies:

Alternative Splice Variants:
The Δ5 STAT5A variant (missing exon 5) retains phosphorylation capability but demonstrates altered interaction with transcription factors and differential effects on target gene expression. This variant:

• Interacts with different transcription factors (AP-1-2, SP1) compared to full-length STAT5A

• Inhibits p21 and Bax production

• Promotes increased cell number

• Is found at higher ratios in invasive ductal carcinoma

Subcellular Localization Differences:
STAT5A function depends on its location:

• Nuclear STAT5A primarily regulates transcription

• Mitochondrial STAT5A interacts with PDC to regulate metabolism

• Cytoplasmic STAT5A may have distinct non-transcriptional functions

Phosphorylation-Independent Functions:
Some STAT5A functions occur independently of tyrosine phosphorylation, as demonstrated in studies with non-phosphorylatable mutants .

Paralog Compensation:
STAT5B may compensate for STAT5A in some contexts, masking functional deficits. Conversely, dominant-negative effects have been observed in some STAT5A mutants .

To resolve contradictory results:

• Assess both phosphorylation status and subcellular localization

• Check for splice variants using appropriate primers/antibodies

• Consider both transcriptional and non-transcriptional functions

• Evaluate the balance between STAT5A and STAT5B expression

What are common artifacts in STAT5A immunodetection and how can they be avoided?

Several artifacts can complicate STAT5A immunodetection:

For phospho-STAT5A detection:

• Rapid sample processing is essential to maintain phosphorylation status

• Include both positive controls (cytokine-stimulated cells) and negative controls (phosphatase-treated samples)

• Verify specificity using phosphatase treatment or phospho-deficient mutants

• Consider basal phosphorylation levels in different cell types

How can STAT5A phosphorylation be utilized as a biomarker for T cell function assessment?

STAT5A phosphorylation provides a rapid and sensitive indicator of T cell activation status, offering advantages over traditional proliferation assays:

Flow Cytometry-Based pSTAT5A Assay:

• Stimulate T cells with CD3/CD28 or PHA

• Fix cells with formaldehyde (2%)

• Permeabilize with methanol (100%)

• Stain with fluorochrome-labeled anti-pY694 STAT5 antibodies

• Co-stain with surface markers for T cell subsets

• Analyze by flow cytometry

Key Advantages:

• Rapid results (hours versus days for proliferation assays)

• Single-cell resolution

• Compatible with small sample volumes

• Allows subset-specific analysis

• High sensitivity for detecting functional defects

This approach has proven valuable for assessing T cell function in:

• Primary immunodeficiencies

• Autoimmune diseases

• CD25 deficiency

• T cell proliferation defects

Research by Fröhlich et al. demonstrated strong correlation between early CD3/CD28-induced STAT5A phosphorylation and subsequent T cell proliferation, validating this as a predictive biomarker .

What approaches can be used to study the role of STAT5A in cancer development and progression?

STAT5A has complex roles in cancer biology, sometimes promoting and other times suppressing tumor growth. Several approaches can elucidate these functions:

Expression and Phosphorylation Analysis:

• Quantify STAT5A protein levels in tumor versus normal tissues

• Assess nuclear versus cytoplasmic localization (transcriptionally active versus inactive)

• Evaluate phosphorylation status

• Examine splice variant ratios (e.g., Δ5 STAT5A versus full-length)

Functional Studies:

• Overexpress constitutively active STAT5A(1*6) mutant to assess oncogenic potential

• Knockdown STAT5A using siRNA/shRNA to evaluate dependency

• Express dominant-negative forms to investigate signaling requirements

• Use selective inhibitors to distinguish STAT5A from STAT5B functions

Protein-Protein Interaction Studies:
STAT5A interactions with:

• Androgen receptor (AR) in prostate cancer

• Vitamin D and retinoid X receptors

• β-catenin

• PDC in mitochondria
  These interactions can be studied using co-immunoprecipitation, proximity ligation assays, or FRET

Research indicates that STAT5A can affect cancer progression through various mechanisms:

• Protection of AR against proteasomal degradation in prostate cancer

• Altered gene expression profiles affecting cell proliferation and survival

• Promotion of epithelial-to-mesenchymal transition (EMT)

How do I design experiments to distinguish between redundant and non-redundant functions of STAT5A and STAT5B?

Despite their high sequence homology, STAT5A and STAT5B exhibit both redundant and unique functions. Designing experiments to distinguish these requires:

Genetic Approaches:

• Use single knockout (KO) models (Stat5a-/- or Stat5b-/-)

• Compare with double knockout heterozygotes (Stat5a+/-Stat5b+/-)

• Generate knock-in (KI) models with tyrosine mutants to create dominant negatives

• Create paralog-specific rescue experiments

Gene Regulation Analysis:

• Perform ChIP-seq using:

  • Paralog-specific antibodies in wild-type cells, or

  • A single antibody recognizing both paralogs in paralog-deficient cells

• Compare genomic distribution patterns

• Correlate with gene expression changes using RNA-seq

• Focus on both common and differentially regulated genes

Protein-Protein Interaction Comparisons:

• Identify differential binding partners for each paralog

• Map interaction domains

• Generate chimeric proteins to determine functional domains

Research using these approaches has revealed:

• Some genes (Enah, Lama5) are specifically affected by one particular STAT5 protein

• IL-2Rα induction requires both STAT5A and STAT5B

• IL-2Rβ regulation varies between IL-2 and IL-3 signaling contexts

• STAT5B is typically more dominant in immune cell functions

• STAT5A has unique roles in mammary tissue

Villarino et al. demonstrated that paralog redundancy varies by context, with STAT5B showing greater dominance in T cells. Their work suggests therapeutic targeting of STAT5A may offer safer but potentially less robust effects than targeting STAT5B .

What are the optimal sample preparation methods for preserving STAT5A phosphorylation status?

Preserving STAT5A phosphorylation is critical for accurate assessment of its activation state:

Cell Culture Samples:

• Stimulate cells with appropriate cytokines/growth factors (IL-2, IL-3, prolactin, etc.)

• Wash briefly with ice-cold PBS

• Lyse directly in buffer containing:

  • Phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Protease inhibitors (PMSF, aprotinin, leupeptin)

  • SDS for immediate denaturation

• Heat samples at 95°C for 5 minutes

• Process immediately or flash-freeze in liquid nitrogen

Tissue Samples:

• Collect tissues with minimal ischemia time

• Immediately snap-freeze in liquid nitrogen or fix appropriately

• For fresh tissue analysis, homogenize directly in lysis buffer with inhibitors

• For fixed tissues, use phospho-specific antibodies validated for IHC

• Consider using phospho-preserving fixatives (e.g., Biocare's Phospho-STAT fixative)

Flow Cytometry Preparation:

• Fix cells with 2% formaldehyde immediately after stimulation

• Permeabilize with 100% methanol

• Stain with phospho-specific antibodies (anti-pY694 STAT5)

Research by Liu et al. demonstrated that immediate processing with phosphatase inhibitors is essential for detecting physiological changes in STAT5A phosphorylation states during cellular responses .

What are the advantages and limitations of different STAT5A detection methods in experimental settings?

Various detection methods offer different strengths and limitations for STAT5A research:

Method Selection Guidance:

• For phosphorylation dynamics: Flow cytometry or Western blotting

• For spatial distribution: Immunofluorescence or IHC

• For protein interactions: Co-IP or proximity ligation assay

• For genomic targets: ChIP-seq or CUT&RUN

• For splice variants: Western blotting or RT-PCR followed by protein analysis

Research by Li et al. used a combination of tandem affinity purification with mass spectrometry to identify novel STAT5A interacting partners, demonstrating the value of integrating multiple technical approaches .