Phospho-PGR (Ser190) Antibody

What is Phospho-Progesterone Receptor (Ser190) and why is it significant in research?

Phospho-Progesterone Receptor (Ser190) refers to the progesterone receptor protein that has been phosphorylated at the serine residue at position 190. The progesterone receptor (PR) is a member of the steroid family of nuclear receptors that mediates the physiological effects of progesterone, playing a central role in reproductive events associated with pregnancy establishment and maintenance. Phosphorylation at Ser190 represents a crucial post-translational modification that regulates PR transcriptional activity and hormone responsiveness. This specific phosphorylation site is significant because it affects receptor stability, nuclear localization, and interaction with transcriptional coregulators, making it an important marker for studying PR function in various physiological and pathological processes .

The progesterone receptor exists in two main isoforms: PR-A (94 kDa) and PR-B (120 kDa). PR-B is the transcriptionally active form that activates genes for endometrium maintenance, pregnancy maintenance, and ovulation inhibition. PR-A is identical to PR-B except for a 165 amino acid deletion at the N-terminus, which exposes an inhibitory domain that acts as a repressor of steroid hormone transcriptional activity . Phosphorylation at Ser190 can differentially affect these isoforms, making it a critical research focus for understanding progesterone signaling mechanisms.

How do polyclonal and monoclonal Phospho-PGR (Ser190) antibodies differ in research applications?

Polyclonal antibodies, such as the rabbit anti-Phospho-Progesterone Receptor (Ser190) antibody, recognize multiple epitopes surrounding the phosphorylated Ser190 site, offering high sensitivity but potentially greater background. These antibodies are typically used at dilutions of 1:500-1:1000 for Western blot and 1:50-1:100 for immunohistochemistry . In contrast, monoclonal antibodies like MA1-413 recognize a single epitope comprising the phosphorylated Ser190 residue, providing higher specificity but potentially lower sensitivity. The monoclonal antibody has been validated for Western blot, immunohistochemistry, ELISA, immunofluorescence, and immunoprecipitation procedures . The choice between these antibody types depends on the specific research question, with monoclonals preferred for precise epitope targeting and polyclonals for enhanced detection sensitivity.

What are the optimal storage and handling conditions for Phospho-PGR (Ser190) antibodies?

For maximum antibody stability and performance, Phospho-Progesterone Receptor (Ser190) antibodies should be stored at -20°C, where they remain stable for at least one year . The antibodies are typically supplied in PBS (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl . Researchers should avoid repeated freeze-thaw cycles, which can degrade antibody quality and reduce specificity and sensitivity.

When handling the antibody:

• Aliquot upon first thaw to minimize freeze-thaw cycles

• Thaw completely at room temperature before use

• Briefly centrifuge before opening the vial to collect all material

• Always maintain sterile conditions when handling

• Return to -20°C immediately after use

For working dilutions, researchers should prepare only the amount needed for immediate use. The recommended dilutions vary by application: 1:500-1:1,000 for immunoblotting, 1:100-1:200 for immunofluorescence, and 1:50-1:100 for immunohistochemistry . These are starting recommendations, and optimal dilutions should be determined empirically for each experimental condition and sample type.

What cell and tissue models are recommended for studying Phospho-PGR (Ser190)?

MCF7 human breast cancer cells represent the gold standard cell model for studying Phospho-Progesterone Receptor (Ser190). These cells express both progesterone receptor isoforms and respond predictably to hormonal stimulation, making them ideal for studying phosphorylation dynamics . In experimental settings, MCF7 cells have demonstrated reliable Phospho-PGR (Ser190) responses to various treatments:

• 17-β Estradiol (0.78 nM, 24 hours) - Increases both phosphorylated and total progesterone receptor levels

• Fulvestrant (10 μg/mL, 18 hours) - Decreases progesterone receptor expression by antagonizing estrogen receptor signaling

• Promegestone/R5020 (1 μM, 2 hours) - Synthetic progestin that induces rapid Ser190 phosphorylation

T47D cells represent another valuable model, particularly for Western blot detection of both PR-A (~81 kDa) and PR-B (~116 kDa) phosphorylated forms . For tissue-based studies, human breast carcinoma samples have shown robust detection of phosphorylated PR . In reproductive biology research, the heterogeneous expression of PR isoforms in uterine tissue makes this an informative model system - isoforms A and B are expressed at comparable levels in uterine glandular epithelium during the proliferative phase of the menstrual cycle, while expression of isoform B (but not A) persists in the glands during the mid-secretory phase .

What is the significance of serine 190 phosphorylation in progesterone receptor function?

Phosphorylation of progesterone receptor at serine 190 represents a crucial regulatory mechanism that modulates receptor activity, localization, and protein-protein interactions. This specific phosphorylation site is located within the sequence G-L-S(p)-P-A of the human progesterone receptor , positioned in a region that influences receptor conformation and function. Serine 190 phosphorylation affects several key aspects of PR biology:

• Transcriptional Activity: Phosphorylation at Ser190 can either enhance or repress PR transcriptional activity depending on cell context and the presence of other post-translational modifications.

• Hormone Sensitivity: This modification modulates the receptor's sensitivity to progesterone and synthetic progestins, as demonstrated in experimental protocols using Promegestone (R5020) .

• Isoform-Specific Effects: Ser190 phosphorylation can differentially affect PR-A and PR-B isoforms, potentially contributing to their distinct functions in tissues like the uterine glandular epithelium during different phases of the menstrual cycle .

• Integration with Other Signaling Pathways: Ser190 phosphorylation is responsive to hormonal treatments beyond progesterone, including estradiol, indicating cross-talk between estrogen and progesterone signaling pathways .

• Disease Relevance: Altered phosphorylation patterns at Ser190 have been implicated in reproductive disorders and hormone-responsive cancers, making this modification a potential biomarker or therapeutic target.

What are the optimal protocols for detecting Phospho-PGR (Ser190) by Western blot?

The detection of Phospho-Progesterone Receptor (Ser190) by Western blot requires careful optimization to ensure specificity and sensitivity. Based on validated protocols, the following methodology is recommended:

Sample Preparation:

• Culture cells to appropriate confluence (e.g., MCF7 cells in T175 flasks)

• Treat cells with appropriate stimuli (e.g., 17-β Estradiol, Fulvestrant, or Promegestone/R5020)

• Harvest cells and lyse using a buffer that preserves phosphorylation (containing phosphatase inhibitors)

• Determine protein concentration and normalize across samples

Western Blot Protocol:

• Separate 20-50 μg of protein on 7.5-10% SDS-PAGE gel (optimal for resolving high molecular weight PR isoforms)

• Transfer proteins to PVDF membrane (preferred over nitrocellulose for phosphorylated proteins)

• Block membrane with 5% BSA in TBST (not milk, which contains phosphoproteins)

• Incubate with primary antibody at 1:500-1:1,000 dilution overnight at 4°C

• Wash thoroughly with TBST (4× 5 minutes)

• Incubate with appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG)

• Develop using enhanced chemiluminescence

Expected Results:

• Polyclonal antibodies typically detect bands at 80-130 kDa

• Monoclonal antibodies precisely identify PR-A at ~81 kDa and PR-B at ~116 kDa

• Positive controls should include MCF7 cell lysates treated with R5020

• Negative controls should include lambda phosphatase-treated samples

To ensure specificity, researchers should validate results by comparing phospho-specific antibody detection with total PR antibody detection, and by using phosphatase treatment to confirm that the signal is phosphorylation-dependent.

How can researchers optimize immunohistochemical detection of Phospho-PGR (Ser190)?

Immunohistochemical detection of Phospho-Progesterone Receptor (Ser190) in tissue sections requires specific protocol adaptations to preserve phosphoepitopes while maintaining tissue morphology. The following optimized protocol is recommended for paraffin-embedded tissue sections:

Tissue Preparation and Antigen Retrieval:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section tissues at 4-5 μm thickness

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes

  • Critical: Add phosphatase inhibitors (e.g., 1 mM sodium orthovanadate) to the retrieval buffer

Staining Protocol:

• Block endogenous peroxidase with 3% hydrogen peroxide

• Apply protein block (e.g., 5% normal goat serum) for 30 minutes

• Incubate with primary antibody at a dilution of 1:50-1:100 overnight at 4°C

  • For polyclonal antibodies: start at 1:50 dilution

  • For monoclonal antibodies: follow manufacturer's recommended dilution

• Wash thoroughly with PBS containing phosphatase inhibitors

• Apply appropriate detection system (e.g., polymer-based detection system)

• Develop with DAB and counterstain with hematoxylin

• Dehydrate, clear, and mount sections

Controls and Validation:

• Positive control: Human breast carcinoma tissue (preferably hormone-responsive)

• Negative controls: (1) Primary antibody omission; (2) Tissue known to be PR-negative

• Validation control: Serial sections stained with total PR antibody

• Phospho-specificity control: Pre-treatment of serial sections with lambda phosphatase

For fluorescent detection, researchers should use 1:100-1:200 dilution of primary antibody and appropriate fluorophore-conjugated secondary antibodies. Nuclear counterstaining with DAPI is recommended for clear visualization of nuclear localization of phosphorylated PR.

How should researchers design controls for Phospho-PGR (Ser190) experiments?

Properly designed controls are essential for ensuring the validity and reliability of Phospho-Progesterone Receptor (Ser190) experiments. A comprehensive control strategy should include:

Positive Controls:

• Cell Line Controls: MCF7 cells treated with 1 μM Promegestone (R5020) for 2 hours show substantial Ser190 phosphorylation

• Tissue Controls: Well-characterized PR-positive breast carcinoma samples

• Lysate Controls: Commercially available positive control lysates prepared from MCF7 cells cultured to confluence in T175 flasks in 10% FBS containing medium

Negative Controls:

• Antibody Omission Control: Sample processed without primary antibody to detect non-specific binding of secondary antibody

• PR-Negative Samples: Cell lines or tissues known to lack PR expression

• Blocking Peptide Control: Primary antibody pre-incubated with the immunizing phosphopeptide (G-L-S(p)-P-A)

Specificity Controls:

• Phosphatase Treatment: Treating duplicate samples with lambda phosphatase to remove phosphate groups, which should eliminate signal if the antibody is truly phospho-specific

• Isoform Controls: Comparing detection in cells expressing predominantly PR-A versus PR-B

• Knockdown/Knockout Controls: Samples with PR knockdown or knockout to confirm signal specificity

Quantitative Controls:

• Loading Controls: Housekeeping proteins (e.g., β-actin, GAPDH) to normalize for total protein loading

• Total PR Control: Parallel detection of total PR to calculate phospho-to-total PR ratio

• Dose Response Controls: Treatment with varying concentrations of stimuli (e.g., 17-β Estradiol) to demonstrate signal proportionality

Treatment Validation Controls:

• Time Course Controls: Samples collected at different time points after stimulation

• Inhibitor Controls: Co-treatment with pathway-specific inhibitors to confirm signaling mechanism

• Antagonist Controls: Inclusion of PR antagonists (e.g., RU486) to demonstrate receptor-specific effects

This comprehensive control strategy ensures that any observed changes in Phospho-PR (Ser190) levels are specific, reproducible, and biologically relevant.

What are the best methods to induce or inhibit Ser190 phosphorylation in experimental models?

Researchers have several validated approaches to modulate Phospho-Progesterone Receptor (Ser190) levels in experimental systems, allowing for mechanistic studies of this post-translational modification:

Methods to Induce Ser190 Phosphorylation:

• Progestin Treatment:

  • Promegestone (R5020) at 1 μM for 2 hours significantly increases Ser190 phosphorylation in MCF7 cells

  • Natural progesterone (100 nM - 1 μM) can be used but may show slower kinetics

• Estrogen Pathway Activation:

  • 17-β Estradiol at 0.78 nM for 24 hours upregulates both phosphorylated and total progesterone receptor in MCF7 cells

  • This approach works through estrogen-mediated upregulation of PR expression

• Cell Cycle Regulators:

  • Cyclin-dependent kinases (particularly CDK2) phosphorylate PR at Ser190

  • Cell synchronization followed by release into S phase can enhance Ser190 phosphorylation

• Growth Factor Signaling:

  • EGF treatment (100 ng/ml, 30 minutes) activates MAPK pathways leading to PR phosphorylation

  • Insulin/IGF-1 pathway activation can similarly induce phosphorylation

Methods to Inhibit Ser190 Phosphorylation:

• Receptor Antagonism:

  • Antiprogestins like RU486 (1-10 μM) block ligand-induced phosphorylation

  • Fulvestrant (10 μg/mL, 18 hours) decreases PR expression by antagonizing estrogen receptor, subsequently reducing phosphorylated PR levels

• Kinase Inhibition:

  • CDK inhibitors (e.g., roscovitine, 25 μM) block Ser190 phosphorylation

  • MEK/ERK pathway inhibitors (e.g., U0126, PD98059) prevent growth factor-induced phosphorylation

• Phosphatase Activation:

  • PP1/PP2A phosphatase activators dephosphorylate PR at multiple sites

  • Okadaic acid treatment can be used to study phosphatase involvement (inverse approach)

• Genetic Approaches:

  • Site-directed mutagenesis of Ser190 to Alanine (S190A) creates a non-phosphorylatable receptor

  • CRISPR/Cas9 genome editing to create endogenous S190A mutations

• siRNA Knockdown:

  • Targeting kinases responsible for Ser190 phosphorylation

  • Targeting PR itself to validate antibody specificity

These experimental approaches should be validated by measuring both phosphorylated and total PR levels to distinguish between effects on phosphorylation versus changes in total protein expression.

How can researchers accurately quantify the ratio of phosphorylated to total progesterone receptor?

Accurate quantification of phosphorylated-to-total progesterone receptor ratio is essential for understanding the dynamics of receptor activation. Several methodological approaches can be employed:

Western Blot-Based Quantification:

• Sequential Immunoblotting:

  • Probe first with phospho-specific antibody

  • Strip and re-probe with total PR antibody

  • Use image analysis software to calculate band intensities

  • Express results as phospho-PR/total PR ratio

  • Normalize to baseline or control condition

• Parallel Gel Analysis:

  • Run duplicate gels with identical samples

  • Probe one with phospho-PR and one with total PR antibody

  • Normalize loading with housekeeping proteins

  • Calculate ratio from normalized values

ELISA-Based Methods:

Researchers can utilize specialized detection kits that simultaneously measure phosphorylated and total PR. Data from MCF7 cells treated with 17-β Estradiol (0.78 nM, 24 hours) demonstrates the utility of this approach, revealing induced expression patterns for both phosphorylated and total receptor populations . Similarly, Fulvestrant treatment (10 μg/mL, 18 hours) showed parallel decreases in both phosphorylated and total PR levels, maintaining a relatively stable phosphorylation ratio .

Immunofluorescence-Based Quantification:

• Dual Immunofluorescence:

  • Co-stain with phospho-PR and total PR antibodies (using different species antibodies)

  • Use confocal microscopy for co-localization analysis

  • Measure mean fluorescence intensity in defined cellular compartments

  • Calculate ratio on a cell-by-cell basis

• High-Content Imaging:

  • Automated image acquisition of multiple fields

  • Segmentation of nuclear regions

  • Quantification of phospho and total PR signals

  • Calculation of ratio across cell populations

Flow Cytometry:

• Fix and permeabilize cells

• Stain with directly conjugated phospho-PR and total PR antibodies

• Analyze ratio on a single-cell basis

• Sort cells based on phosphorylation status for further analysis

For all methods, it's critical to include appropriate controls, particularly samples treated with phosphatases to establish baseline (non-phosphorylated) signal levels. Data should be presented as fold-change relative to a defined baseline condition to account for inter-experimental variability.

How should researchers interpret multiple bands on Western blots with Phospho-PGR (Ser190) antibodies?

When using Phospho-Progesterone Receptor (Ser190) antibodies for Western blotting, researchers often encounter multiple bands that require careful interpretation. The pattern of bands provides important information about PR isoforms, degradation products, and potential cross-reactivity:

Expected Band Patterns:

• Major PR Isoforms:

  • PR-B: ~116-120 kDa band representing the full-length receptor

  • PR-A: ~81-94 kDa band representing the N-terminally truncated isoform

  • Both isoforms should be detected by phospho-specific antibodies when phosphorylated at Ser190

• Cell Line-Specific Patterns:

  • T47D and MCF7 cells typically express both PR-A and PR-B isoforms

  • The ratio between isoforms may vary depending on culture conditions and hormonal status

  • Some experimental cell lines may predominantly express one isoform

• Tissue-Specific Patterns:

  • In uterine tissue, heterogeneous expression patterns exist between the glands of the endometrium basalis and functionalis, with isoform A predominant in stroma throughout the menstrual cycle

  • These natural variations should be considered when interpreting tissue sample results

Interpreting Additional Bands:

Troubleshooting Approaches:

• For multiple high molecular weight bands: Ensure complete denaturation by increasing SDS concentration, boiling time, or adding reducing agents

• For degradation products: Add additional protease inhibitors during sample preparation and keep samples cold throughout processing

• For validation: Compare band patterns with a total PR antibody on parallel samples to confirm identity of phosphorylated bands

• For ambiguous results: Perform immunoprecipitation with total PR antibody followed by Western blot with phospho-specific antibody to confirm identity

What are common causes of weak or absent signal when detecting Phospho-PGR (Ser190)?

When researchers encounter weak or absent signals in Phospho-Progesterone Receptor (Ser190) detection experiments, several factors may be responsible. Identifying and addressing these issues is crucial for successful experimental outcomes:

Pre-analytical Factors:

• Phosphoepitope Loss:

  • Phosphorylation sites are extremely labile and sensitive to phosphatase activity

  • Solution: Add phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) to all buffers

  • Process samples quickly and maintain cold temperatures throughout

• Low PR Expression:

  • PR expression is hormone-dependent and varies across cell lines and tissues

  • Solution: Verify PR expression using total PR antibodies before phospho-specific detection

  • Consider hormone pre-treatment (e.g., estradiol) to induce PR expression

• Insufficient Phosphorylation:

  • Baseline phosphorylation at Ser190 may be low without stimulation

  • Solution: Treat cells with Promegestone (R5020, 1 μM for 2 hours) to induce phosphorylation

  • Use positive control lysates from stimulated MCF7 cells

Analytical Factors:

• Antibody-Related Issues:

  • Suboptimal antibody dilution

  • Solution: Titrate antibody concentrations (1:500-1:1000 for WB; 1:50-1:100 for IHC)

  • Consider lot-to-lot variability; validate new antibody lots

• Detection Method Sensitivity:

  • Standard ECL may be insufficient for low-abundance phosphoproteins

  • Solution: Use high-sensitivity detection reagents or amplification systems

  • Increase exposure time while monitoring background

• Blocking Interference:

  • Milk contains phosphoproteins and phosphatases that can interfere with detection

  • Solution: Use BSA instead of milk for blocking and antibody dilution

Post-analytical Factors:

• Interpretation Challenges:

  • Signal may be present but misinterpreted due to molecular weight shifts

  • Solution: Include molecular weight markers and positive controls

  • Compare band patterns with published literature for your cell type

• Quantification Limitations:

  • Low signal-to-noise ratio affecting accurate quantification

  • Solution: Use digital imaging systems with broader dynamic range

  • Apply background subtraction appropriately

For comprehensive troubleshooting, researchers should systematically evaluate each step of their protocol, beginning with validation of total PR expression and proceeding to verification of phosphorylation induction, before addressing technical aspects of detection.

How can researchers distinguish between specific and non-specific binding in Phospho-PGR (Ser190) experiments?

Distinguishing between specific and non-specific binding is crucial for accurate interpretation of Phospho-Progesterone Receptor (Ser190) experiments. Multiple validation approaches should be employed to ensure signal specificity:

Validation Approach 1: Phosphatase Treatment

Phosphatase treatment provides the gold standard for validating phospho-specific antibodies:

• Divide your sample into two aliquots

• Treat one aliquot with lambda phosphatase to remove phosphate groups

• Process both samples identically for detection

• The phosphatase-treated sample should show significantly reduced or absent signal

• Persistent signal after phosphatase treatment indicates non-specific binding

Validation Approach 2: Competing Peptide Controls

The immunizing peptide containing phosphorylated Ser190 (G-L-S(p)-P-A) can be used to validate specificity:

• Pre-incubate primary antibody with excess phosphopeptide

• In parallel, pre-incubate with non-phosphorylated peptide or unrelated peptide

• Apply to duplicate samples

• Specific binding will be blocked by the phosphopeptide but not by control peptides

Validation Approach 3: Genetic Approaches

Genetic manipulation provides definitive evidence of specificity:

• Compare signal in PR-positive versus PR-negative/knockdown cells

• Express wild-type PR versus S190A mutant (non-phosphorylatable)

• Specific phospho-antibodies will detect wild-type but not mutant PR

Validation Approach 4: Signal Characteristics Analysis

Pattern recognition can help distinguish specific from non-specific signals:

Validation Approach 5: Cross-Method Validation

Confirm findings using multiple detection techniques:

• Compare Western blot results with immunohistochemistry or immunofluorescence

• Validate with orthogonal methods such as mass spectrometry

• Specific signals should be consistent across methodologies, while non-specific binding often varies

By implementing these validation approaches systematically, researchers can confidently distinguish between specific Phospho-PGR (Ser190) signal and artifacts or non-specific binding.

How do different detection methods for Phospho-PGR (Ser190) compare in sensitivity and specificity?

Different detection methods for Phospho-Progesterone Receptor (Ser190) offer varying advantages in terms of sensitivity, specificity, and information content. Understanding these differences helps researchers select the most appropriate method for their specific research question:

Method-Specific Considerations:

• Western Blot:

  • Advantages: Distinguishes PR-A (~81-94 kDa) from PR-B (~116-120 kDa) ; confirms specificity by molecular weight

  • Limitations: Limited spatial information; semi-quantitative; requires substantial sample amount

  • Optimization: Use gradient gels (7.5-10%) for optimal isoform separation; highly sensitive ECL substrates

• Immunohistochemistry/Immunofluorescence:

  • Advantages: Preserves tissue architecture; reveals heterogeneity; shows subcellular localization

  • Limitations: Epitope masking in fixed tissues; subjective quantification; background autofluorescence

  • Optimization: Heat-induced epitope retrieval with phosphatase inhibitors; signal amplification systems

• ELISA-Based Detection:

  • Advantages: Highly quantitative; reproducible; amenable to high-throughput

  • Limitations: Loses isoform differentiation; no spatial information; potential cross-reactivity

  • Optimization: Validated kits show differential response to treatments like 17-β Estradiol and Fulvestrant

• Phospho-Flow Cytometry:

  • Advantages: Single-cell resolution; multi-parameter analysis; population statistics

  • Limitations: Complex optimization; fixation affects epitope recognition; limited subcellular information

  • Optimization: Alcohol-based fixation often superior for phospho-epitopes; careful titration of antibodies

• Proximity Ligation Assay (PLA):

  • Advantages: Extremely high sensitivity; detects protein-protein interactions in situ

  • Limitations: Complex protocol; specialized reagents; challenging quantification

  • Optimization: Ideal for studying PR phosphorylation in context of cofactor recruitment

The optimal approach often involves combining multiple methods - for example, validating Western blot findings with immunofluorescence to confirm subcellular localization, or supplementing immunohistochemistry with quantitative ELISA data.

What factors affect the phospho-to-total PGR ratio and how should fluctuations be interpreted?

The phospho-to-total progesterone receptor ratio provides crucial information about receptor activation status, but multiple factors influence this measurement and must be considered during interpretation:

Physiological Factors Affecting Phospho-to-Total Ratio:

• Hormonal Status:

  • Progesterone and synthetic progestins (e.g., R5020) rapidly induce Ser190 phosphorylation without immediate changes in total PR, transiently increasing the phospho/total ratio

  • Prolonged progesterone exposure typically leads to receptor downregulation, potentially decreasing both phosphorylated and total PR

• Cell Cycle Phase:

  • PR phosphorylation fluctuates during cell cycle progression

  • S-phase typically shows enhanced phosphorylation at Ser190 due to CDK2 activity

  • Interpretation must consider cell synchronization status

• Cross-Talk with Other Signaling Pathways:

  • Estrogen signaling increases total PR expression while maintaining phosphorylation, potentially decreasing the phospho/total ratio

  • Growth factor signaling (EGF, IGF-1) can increase phosphorylation without affecting total PR levels

  • Stress signaling pathways may differentially affect phosphorylation versus expression

Experimental Factors Affecting Ratio Measurements:

• Antibody Affinity Differences:

  • Phospho-specific and total PR antibodies may have different affinities

  • Solution: Generate standard curves for both antibodies using purified proteins

  • Express results as relative rather than absolute ratios

• Epitope Accessibility:

  • Phosphorylation may alter epitope accessibility for total PR antibodies

  • Solution: Use total PR antibodies targeting regions distant from phosphorylation sites

  • Validate with multiple total PR antibodies targeting different epitopes

• Detection Method Linearity:

  • Signal saturation can compress apparent differences

  • Solution: Ensure detection is in the linear range for both phospho and total signals

  • Perform serial dilutions to confirm linearity

Interpreting Ratio Changes:

The most informative approach is to report not only the ratio but also the absolute values of both phosphorylated and total PR, allowing for comprehensive interpretation of receptor regulation dynamics.

How can Phospho-PGR (Ser190) analysis contribute to breast cancer research?

Phospho-Progesterone Receptor (Ser190) analysis provides valuable insights into breast cancer biology, potentially informing diagnosis, prognosis, and therapeutic strategies:

Diagnostic Applications:

Phospho-PGR (Ser190) detection offers advantages beyond traditional PR status assessment:

• May identify functionally active PR even in tumors classified as PR-low by conventional IHC

• Could help resolve equivocal PR status by providing functional information

• Potentially distinguishes between active and inactive PR signaling in heterogeneous tumor samples

Prognostic Significance:

Emerging research suggests that phosphorylation status may have prognostic value:

• Altered phosphorylation patterns may identify aggressive tumor phenotypes

• The ratio of phosphorylated to total PR could serve as a biomarker of functional PR signaling

• Specific phosphorylation signatures might predict response to endocrine therapies

Mechanistic Research Applications:

Phospho-PGR (Ser190) analysis facilitates mechanistic studies of breast cancer biology:

• Evaluating cross-talk between estrogen and progesterone signaling, as demonstrated by experiments showing that 17-β Estradiol (0.78 nM, 24 hours) increases both phosphorylated and total progesterone receptor in MCF7 cells

• Understanding the differential effects of selective estrogen receptor modulators/degraders, as shown by studies with Fulvestrant (10 μg/mL, 18 hours)

• Investigating the mechanisms of antiprogestin resistance through phosphorylation-mediated receptor activation

• Studying isoform-specific functions of PR-A (~81 kDa) and PR-B (~116 kDa) in breast cancer progression

Therapeutic Implications:

Phosphorylation status may influence therapeutic strategies:

• Tumors with highly phosphorylated PR might respond differently to progestin therapy

• Targeting kinases responsible for PR phosphorylation could represent a novel therapeutic approach

• Phosphorylation status might predict sensitivity to CDK inhibitors that affect PR phosphorylation

• Combined targeting of PR and its phosphorylation pathways could enhance therapeutic efficacy

Experimental Models and Approaches:

For breast cancer research, established models include:

• MCF7 and T47D cell lines for in vitro studies

• Patient-derived xenografts for translational research

• Human breast carcinoma tissue samples for clinical correlation

Experimental approaches should combine:

• Phospho-specific Western blot to distinguish PR-A and PR-B isoform phosphorylation

• Immunohistochemistry for spatial context in heterogeneous tumors

• Functional assays to correlate phosphorylation with transcriptional activity

• Clinical outcome correlation to establish prognostic relevance

What insights can chromatin immunoprecipitation (ChIP) with Phospho-PGR (Ser190) antibodies provide?

Chromatin immunoprecipitation (ChIP) using Phospho-Progesterone Receptor (Ser190) antibodies offers unique insights into the genomic actions of activated progesterone receptor, revealing how phosphorylation influences receptor-DNA interactions and transcriptional regulation:

Technical Considerations for Phospho-PGR ChIP:

• Protocol Modifications:

  • Include phosphatase inhibitors (10 mM sodium fluoride, 1 mM sodium orthovanadate) in all buffers

  • Shorten crosslinking time (7-10 minutes) to preserve phosphoepitopes

  • Optimize sonication conditions for complete chromatin fragmentation while preserving phosphorylation

  • Use phospho-specific antibodies at higher concentrations than standard ChIP (approximately 5-10 μg per reaction)

• Controls:

  • Parallel ChIP with total PR antibody to normalize for total PR occupancy

  • IgG control to establish background

  • Input chromatin for normalization

  • Phosphatase-treated samples as negative controls for phospho-specificity

Scientific Applications:

• Differential Gene Regulation:
  ChIP-seq with phospho-specific antibodies can identify genomic targets preferentially bound by phosphorylated PR versus total PR, revealing:

  • Unique binding sites of phosphorylated receptor

  • Differential binding strength at shared sites

  • Genomic redistribution following kinase activation

• Transcription Factor Cooperation:
  Phosphorylation at Ser190 influences PR interactions with other transcription factors:

  • Altered tethering to AP-1, Sp1, or NF-κB sites

  • Modified cooperation with estrogen receptor at composite elements

  • Changes in pioneer factor recruitment (e.g., FOXA1)

• Chromatin Remodeling:
  Phosphorylated PR may differentially affect chromatin structure:

  • Recruitment of specific histone modifiers

  • Alterations in chromatin accessibility (can be paired with ATAC-seq)

  • Changes in three-dimensional chromatin organization

• Isoform-Specific Genomic Actions:
  Phospho-PGR ChIP can distinguish between PR-A and PR-B genomic functions:

  • Different phosphorylation-dependent binding patterns between PR-A (~81 kDa) and PR-B (~116 kDa)

  • Isoform-specific coregulator recruitment influenced by phosphorylation

  • Differential response to hormonal stimulation between isoforms

Data Interpretation Framework:

By correlating phospho-PR binding with gene expression data, researchers can establish direct links between Ser190 phosphorylation and specific transcriptional outcomes, providing mechanistic insights into progesterone signaling.

How does Phospho-PGR (Ser190) interact with other post-translational modifications of the progesterone receptor?

Progesterone receptor function is regulated by a complex interplay of multiple post-translational modifications (PTMs), with phosphorylation at Ser190 representing one component of this regulatory network. Understanding these interactions is crucial for comprehending PR function in different cellular contexts:

Interplay with Other Phosphorylation Sites:

Ser190 phosphorylation functions within a network of PR phosphorylation sites:

Experimental approaches to study phosphorylation crosstalk include:

• Sequential immunoblotting with multiple phospho-specific antibodies

• Phospho-mimetic mutations (S→D or S→E) to simulate constitutive phosphorylation

• Mass spectrometry to identify co-occurring phosphorylation patterns

Interactions with Other Types of PTMs:

Ser190 phosphorylation interacts with diverse post-translational modifications:

• Acetylation:

  • Acetylation at K638/K641 may enhance Ser190 phosphorylation

  • Acetylation state affects recruitment of kinases that target Ser190

  • HDAC inhibitors can indirectly increase Ser190 phosphorylation

• SUMOylation:

  • SUMOylation at K388 restricts PR transcriptional activity

  • Ser190 phosphorylation may antagonize SUMOylation

  • The phospho/SUMO switch affects recruitment of coregulators

• Ubiquitination:

  • Ser190 phosphorylation can affect receptor stability by modulating ubiquitination

  • Phosphorylated PR may show altered proteasomal degradation kinetics

  • Proteasome inhibitors can be used to study this relationship

• Methylation:

  • Arginine methylation by PRMT1 affects PR activity

  • Potential crosstalk between methylation and Ser190 phosphorylation remains understudied

Experimental Approaches for Studying PTM Crosstalk:

• Sequential and Orthogonal Immunoprecipitation:

  • First IP with phospho-Ser190 antibody

  • Second IP with antibodies against other PTMs

  • Identifies subpopulations with multiple modifications

• Multi-PTM Mass Spectrometry:

  • Immunoprecipitate PR from hormone-treated cells

  • Analyze by mass spectrometry for combinations of PTMs

  • Quantify relative abundance of different PTM combinations

• Proximity Ligation Assays (PLA):

  • In situ detection of closely associated PTMs

  • Can visualize PTM co-occurrence in different cellular compartments

  • Useful for rare modifications that may be lost in biochemical analyses

• Engineered PR Variants:

  • Generate PR with mutations at multiple PTM sites

  • Compare single mutations vs. combined mutations

  • Assess functional outcomes such as transcriptional activity, subcellular localization, and protein-protein interactions

Understanding this PTM network is crucial for developing a comprehensive model of PR regulation and may inform therapeutic strategies targeting specific aspects of PR signaling.

What are emerging applications of Phospho-PGR (Ser190) detection in reproductive medicine?

Phospho-Progesterone Receptor (Ser190) detection is opening new avenues for research and potential clinical applications in reproductive medicine, building on our understanding of PR isoform expression patterns in reproductive tissues:

Endometrial Receptivity Assessment:

The human endometrium undergoes dynamic changes in PR expression and phosphorylation during the menstrual cycle:

• PR isoforms A and B are expressed at comparable levels in uterine glandular epithelium during the proliferative phase

• Expression of isoform B (but not A) persists in the glands during mid-secretory phase

• In the stroma, isoform A is the predominant form throughout the cycle

Emerging applications include:

• Analysis of Ser190 phosphorylation as a potential biomarker of endometrial receptivity

• Correlation of phosphorylation patterns with successful implantation outcomes

• Development of minimally invasive endometrial sampling techniques for phospho-PR assessment

Recurrent Pregnancy Loss Investigation:

Aberrant progesterone signaling may contribute to recurrent pregnancy loss, with phosphorylation status providing insights beyond conventional PR expression analysis:

• Assessment of phospho-PR/total PR ratio in decidual tissue

• Correlation of abnormal phosphorylation patterns with pregnancy outcomes

• Potential therapeutic approaches targeting phosphorylation pathways

Endometriosis and Adenomyosis:

These conditions involve progesterone resistance that may be linked to altered PR phosphorylation:

• Comparison of eutopic vs. ectopic endometrium for phospho-PR patterns

• Investigation of kinase/phosphatase imbalances affecting Ser190 phosphorylation

• Development of targeted therapies addressing phosphorylation-mediated progesterone resistance

Preterm Birth Risk Assessment:

Progesterone receptor function in myometrium is critical for maintaining uterine quiescence:

• Evaluation of myometrial phospho-PR status across gestation

• Investigation of phosphorylation changes preceding labor onset

• Potential development of phospho-PR-based biomarkers for preterm labor risk

Assisted Reproductive Technology Optimization:

Luteal phase support is critical in assisted reproduction:

• Correlation of endometrial phospho-PR status with implantation success

• Personalization of progesterone supplementation based on receptor phosphorylation profile

• Optimization of stimulation protocols to enhance appropriate PR phosphorylation

Methodology Adaptations for Reproductive Tissues:

• Tissue-Specific Protocols:

  • Fixation optimization for endometrial biopsies to preserve phosphoepitopes

  • Decalcification protocols compatible with phospho-PR detection in bone tissues for reproductive cancer studies

  • Micro-dissection techniques to study heterogeneous expression between endometrial compartments

• Specialized Applications:

  • In situ proximity ligation assays to study PR isoform-specific phosphorylation in intact tissues

  • Laser capture microdissection combined with phospho-PR analysis for region-specific assessment

  • Single-cell analysis techniques to address cellular heterogeneity in reproductive tissues

These emerging applications highlight the potential for phospho-PR analysis to provide functional insights beyond conventional PR expression studies, potentially transforming our understanding of progesterone action in reproductive physiology and pathology.

How can Phospho-PGR (Ser190) detection be adapted for high-throughput screening applications?

Adapting Phospho-Progesterone Receptor (Ser190) detection for high-throughput screening (HTS) enables large-scale investigation of compounds or genetic factors that modulate PR phosphorylation. This approach requires specific methodological considerations to ensure reliability, reproducibility, and efficiency:

Assay Platform Options:

• ELISA-Based Platforms:

  • AlphaLISA or similar bead-based technologies offer high sensitivity with minimal sample requirements

  • Commercial detection kits for Phospho-PGR (Ser190) can be adapted to 384-well formats

  • Homogeneous assays (no-wash) increase throughput and reduce variability

  • Demonstration data shows reliable detection of changes induced by 17-β Estradiol, Fulvestrant, and Promegestone

• High-Content Imaging:

  • Automated immunofluorescence in microplate format

  • Simultaneous detection of phospho-PR and total PR

  • Additional parameters (nuclear translocation, aggregation, colocalization)

  • Machine learning algorithms for complex phenotype analysis

• In-Cell Western Assays:

  • Infrared dye-labeled secondary antibodies for quantitative detection

  • Dual-channel detection of phospho-PR and total PR

  • Normalization to cell number using DNA dyes

  • Suitable for both adherent and suspension cell types

Assay Development Considerations:

Quality Control Measures:

• Plate Controls:

  • Maximum signal: R5020-treated cells (1 μM, 2 hours)

  • Minimum signal: Lambda phosphatase-treated lysates

  • Reference compound: 17-β Estradiol at defined concentration (0.78 nM)

  • Vehicle controls: Positioned strategically to detect position effects

• Assay Validation:

  • Dose-response curves with known modulators

  • Reproducibility assessment (inter-plate, inter-day)

  • Edge effects evaluation

  • DMSO tolerance testing for compound screening

Applications in Drug Discovery:

• Target Classes:

  • Kinase inhibitors affecting PR phosphorylation

  • Novel PR modulators with distinct phosphorylation profiles

  • Compounds affecting PR-kinase interactions

  • Indirect modulators of PR phosphorylation (e.g., via estrogen signaling)

• Screening Approaches:

  • Primary screens: single concentration, phospho/total ratio

  • Confirmation: dose-response, multiple time points

  • Counter-screens: cytotoxicity, general phosphorylation effects

  • Mechanism elucidation: pathway component inhibitors

• Translational Extensions:

  • Patient-derived cells for personalized medicine approaches

  • Correlation of ex vivo response with clinical outcomes

  • Identification of biomarkers predicting drug sensitivity

High-throughput phospho-PR detection represents a powerful approach for identifying novel modulators of progesterone signaling with potential applications in reproductive medicine, endocrine disorders, and hormone-responsive cancers.

Future Directions in Phospho-PGR (Ser190) Research

The field of progesterone receptor phosphorylation research continues to evolve, with several promising directions:

• Single-Cell Analysis: Emerging technologies will enable phospho-PR detection at the single-cell level, revealing heterogeneity in PR signaling within tissues, particularly important given the heterogeneous expression patterns observed between the glands of the endometrium basalis and functionalis .

• Systems Biology Approaches: Integration of phospho-PR data with other -omics platforms (transcriptomics, proteomics, metabolomics) will provide comprehensive models of progesterone signaling networks and their dysregulation in disease states.

• Therapeutic Development: Targeting kinases or phosphatases that regulate Ser190 phosphorylation represents a novel approach to modulating PR function with potential applications in contraception, fertility enhancement, and cancer treatment.

• Biomarker Development: Phospho-PR/total PR ratios may serve as functional biomarkers of progesterone signaling activity in tissues, potentially improving prediction of therapeutic response in hormone-dependent conditions.

• Methodological Advances: Development of more sensitive and specific detection methods, including advanced proximity-based assays and highly multiplexed imaging approaches, will enhance our ability to study PR phosphorylation in complex tissue environments.