Phospho-AR (Ser213) Antibody

What is Phospho-AR (Ser213) and why is it significant in prostate cancer research?

Phospho-AR (Ser213) refers to the androgen receptor (AR) that is specifically phosphorylated at the serine 213 residue. This specific phosphorylation has significant implications for prostate cancer research because it represents a putative substrate for Akt (protein kinase B), directly linking AR function to the PI3K/Akt signaling pathway . Studies have demonstrated that the phosphorylation at this site occurs with rapid kinetics in response to androgens like R1881 and dihydrotestosterone, but weakly if at all with testosterone .

AR Ser213 phosphorylation shows cell-type specific patterns, being present in prostate epithelial cells but notably absent in stromal cells despite AR expression in both cell types . This phosphorylation may play a critical role in the progression of prostate cancer to an androgen-insensitive state, with increases in both phospho-Akt and phospho-AR Ser213 found in castration-resistant prostate cancer compared to hormone-naïve tumors .

Interestingly, research has shown that while there is a synergy between AKT and AR signaling that can transform naïve prostatic epithelium into androgen-insensitive carcinoma, phosphorylation of AR at Ser-213 and Ser-791 by AKT is not critical for this synergy . This suggests complex mechanisms beyond direct phosphorylation in the interaction between these pathways.

How are Phospho-AR (Ser213) antibodies validated for research applications?

Validation of Phospho-AR (Ser213) antibodies requires multiple complementary approaches to ensure specificity and reliability for research applications:

• Peptide specificity confirmation: Antibodies are generated and validated against synthetic phosphopeptides containing the phosphorylated Ser213 sequence (E-A-S(p)-G-A) derived from the human androgen receptor .

• Mutational analysis validation: AR mutants where Ser213 is substituted with alanine (S213A) serve as critical negative controls, as they should not be recognized by a truly phospho-specific antibody .

• Cell line validation: Specific cell lines serve as standard positive controls, with DU145 cells recommended for Western blot applications and HeLa cells for immunocytochemistry/immunofluorescence applications .

• Phosphatase treatment controls: Samples treated with phosphatases should show reduced or absent signal, confirming the phospho-specificity of the antibody.

• Cell type specificity verification: Validation includes testing against cells known to differentially regulate AR Ser213 phosphorylation, such as prostate epithelial versus stromal cells .

• Pathway inhibitor confirmation: Treatment with PI3K inhibitors like LY294002 should reduce signal, confirming the antibody's ability to detect phosphorylation dependent on this pathway .

A properly validated Phospho-AR (Ser213) antibody should demonstrate high specificity for AR phosphorylated at Ser213 with minimal cross-reactivity to unphosphorylated AR or AR phosphorylated at other residues.

How does phosphorylation at AR Ser213 affect androgen receptor function?

Phosphorylation of AR at Ser213 has complex and context-dependent effects on receptor function:

Transcriptional activity regulation: Activated PI3K/Akt pathway inhibits transcription mediated by wild-type AR but not that of the S213A mutant AR variant (which cannot be phosphorylated at Ser213), suggesting that phosphorylation at this site may suppress AR transcriptional activity in certain contexts .

Cell-specific regulation patterns: AR Ser213 phosphorylation displays remarkable cell-type specificity, being detected in prostate epithelial cells but not in stromal cells despite AR expression in both cell types . This suggests cell-specific regulatory mechanisms and potentially distinct functional outcomes.

Developmental regulation: In fetal tissue, AR-Ser(P)-213 immunoreactivity is present in differentiated cells lining the lumen of the urogenital sinus but absent in rapidly dividing, Ki67-positive cells within the developing prostate . This pattern suggests that site-specific phosphorylation of AR Ser213 occurs predominantly in non-proliferating, differentiated cellular environments.

Integration with signaling pathways: While phosphorylation at Ser213 is mediated by Akt, the synergy between AKT and AR signaling in promoting androgen-insensitive prostate cancer does not critically depend on this specific phosphorylation . This indicates complex interactions between these pathways beyond direct phosphorylation.

Role in treatment resistance: Increased levels of both phospho-Akt and phospho-AR Ser213 have been observed in castration-resistant prostate cancer compared to hormone-naïve tumors , suggesting potential involvement in treatment resistance mechanisms.

These findings demonstrate that phosphorylation at Ser213 likely serves as a regulatory switch for AR function, with implications for normal development, cancer progression, and therapeutic response.

What experimental methodologies are most effective for studying AR Ser213 phosphorylation dynamics?

Multiple complementary experimental approaches can be employed to comprehensively study AR Ser213 phosphorylation dynamics:

Genetic approaches:

• Site-directed mutagenesis creating S213A mutants (cannot be phosphorylated) to study functional consequences of phosphorylation loss

• Lentiviral expression systems for stable expression of wild-type or mutant AR in cellular models

• RNA interference targeting kinases involved in Ser213 phosphorylation (e.g., Akt)

Biochemical approaches:

• Western blotting with phospho-specific antibodies for detection and relative quantification

• Mass spectrometry for absolute confirmation of phosphorylation sites and stoichiometry

• Reversed-phase HPLC for separation and analysis of different AR phospho-isoforms

• Immunoprecipitation to isolate AR complexes and study associated proteins

Cellular and tissue approaches:

• Immunohistochemistry to detect cell-specific phosphorylation patterns in tissues

• Immunofluorescence microscopy for subcellular localization of phosphorylated AR

• Proximity ligation assays to detect interactions between phospho-AR and binding partners

• FRET/BRET-based approaches to study real-time phosphorylation dynamics

Functional assessment approaches:

• Transcriptional reporter assays using AR-responsive constructs to assess how Ser213 phosphorylation affects AR activity

• Chromatin immunoprecipitation to determine genomic binding sites of phosphorylated AR

• Cell proliferation and survival assays to assess biological consequences

• Drug response studies to evaluate therapeutic implications

Pathway perturbation approaches:

• Kinase inhibitor studies using PI3K/Akt pathway inhibitors like LY294002

• Hormone stimulation assays with androgens (R1881, DHT) to induce AR phosphorylation

• Growth factor treatments to assess cross-talk with AR signaling pathways

The most robust experimental designs combine multiple approaches to provide complementary insights into both the regulation and functional significance of AR Ser213 phosphorylation.

How do cell-specific contexts influence AR Ser213 phosphorylation patterns?

AR Ser213 phosphorylation exhibits striking cell-type specific regulation patterns that provide insights into its biological roles:

Cell lineage-specific regulation:

Prostate epithelial cells consistently show detectable phosphorylation at AR Ser213 when AR is present . In contrast, prostate stromal cells express AR but show minimal or no phosphorylation at Ser213 . This dichotomy suggests fundamental differences in AR regulation between these cell lineages, possibly related to differences in Akt activation or phosphatase activity.

Developmental context-dependent regulation:

In fetal tissue, AR-Ser(P)-213 immunoreactivity is present in epithelial cells of the urogenital sinus when endogenous androgen levels are high and activated Akt is prevalent, but absent at later developmental stages when androgen levels decline and Akt activation diminishes .

Proliferation status-dependent regulation:

AR Ser213 phosphorylation shows inverse correlation with proliferative status. It is detected in differentiated cells lining the lumen of the urogenital sinus but notably absent in rapidly dividing, Ki67-positive cells within the developing prostate and stromal tissue . This pattern suggests that this phosphorylation event occurs predominantly in non-proliferating cellular environments and may be associated with differentiation rather than proliferation.

Malignancy-associated changes:

Prostate cancer cells frequently exhibit increased phosphorylation at Ser213 compared to normal prostate cells, with particularly elevated levels in castration-resistant prostate cancer . This suggests that cancer progression may disrupt normal phosphorylation regulatory mechanisms.

Hormone responsiveness correlation:

AR Ser213 phosphorylation occurs robustly in response to R1881 and dihydrotestosterone but weakly if at all with testosterone , indicating hormone-specific regulation of this phosphorylation site.

These cell-specific regulatory patterns provide important contextual understanding for interpreting experimental results and may guide therapeutic approaches targeting AR signaling in prostate cancer.

What are the optimal Western blotting protocols for Phospho-AR (Ser213) detection?

Successful Western blotting for Phospho-AR (Ser213) requires meticulous attention to phospho-epitope preservation and specificity:

Sample preparation:

• Harvest cells/tissues in ice-cold lysis buffer containing both protease inhibitors and phosphatase inhibitor cocktails

• Maintain samples at 4°C throughout processing to minimize phosphatase activity

• Standardize protein concentration to 1-2 μg/μL for consistent loading

• Include DU145 cell lysates as positive controls for Phospho-AR (Ser213)

Gel electrophoresis parameters:

• Use 7.5-8% SDS-PAGE gels for optimal resolution of AR (~110-114 kDa)

• Load 20-40 μg total protein per lane

• Include molecular weight markers spanning 75-150 kDa range

• Consider running multiple samples of the same lysate for technical replication

Membrane transfer conditions:

• Use wet transfer with 10% methanol for efficient transfer of large proteins

• Transfer at 30V overnight at 4°C for complete transfer of high molecular weight proteins

• Verify transfer efficiency with reversible protein stains before blocking

Antibody incubation parameters:

• Block membranes in 5% BSA in TBS-T (not milk, which contains phosphatases)

• Use 1:1000 dilution of Phospho-AR (Ser213) antibody in blocking buffer

• Incubate primary antibody overnight at 4°C with gentle rocking

• Wash extensively with TBS-T (at least 3 × 10 minutes)

• Use high-sensitivity HRP-conjugated anti-rabbit secondary antibody (1:5000 dilution)

Signal detection considerations:

• Use enhanced chemiluminescence with optimized exposure times

• Consider stripping and reprobing with total AR antibody for normalization

• Include ERK2 as loading control as established in published research

Critical validation controls:

Following these optimized protocols will maximize sensitivity and specificity for detecting AR phosphorylated specifically at Ser213.

How should researchers quantify Phospho-AR (Ser213) levels in experimental systems?

Reliable quantification of AR Ser213 phosphorylation requires rigorous methodology and appropriate normalization:

Western blot-based quantification:

• Normalize phospho-AR (Ser213) signal to total AR signal from the same samples to account for variations in total AR expression

• Use digital imaging systems with wide dynamic range to ensure signal linearity

• Perform densitometric analysis using ImageJ or similar software with background subtraction

• Include a dilution series of positive control sample to confirm signal linearity

• Always report the phospho-AR/total-AR ratio rather than absolute phospho-AR values

• Include ERK2 as additional loading control

Immunohistochemistry quantification approaches:

• Use digital pathology software for unbiased quantification when possible

• Have samples scored by multiple observers blinded to sample identity

• Include both normal and cancer tissues within the same slide as internal references

• Consider dual staining for total AR to calculate phosphorylation percentage

ELISA-based methods:

• Develop sandwich ELISAs using capture antibodies against AR and detection with phospho-specific antibodies

• Include recombinant phosphorylated AR protein or peptide standards for absolute quantification

• Normalize to total AR levels measured in parallel assays

Mass spectrometry approaches:

• Use multiple reaction monitoring (MRM) for targeted quantification of specific phosphopeptides

• Incorporate isotope-labeled synthetic phosphopeptides as internal standards

• Calculate stoichiometry by comparing phosphopeptide to non-phosphopeptide ratios

• Consider HPLC separation of AR isoforms before analysis

Statistical considerations:

• Always perform at least three independent biological replicates

• Use appropriate statistical tests based on data distribution

• Report both absolute and relative changes in phosphorylation

• Consider multivariate analysis when examining multiple phosphorylation sites

These quantification approaches provide complementary information and should be selected based on the specific research question and available resources.

What controls are essential when using Phospho-AR (Ser213) antibodies in research?

Comprehensive controls are critical for reliable interpretation of results with Phospho-AR (Ser213) antibodies:

Positive controls:

• Cell line controls: DU145 cells for Western blot and HeLa cells for immunocytochemistry as recommended for commercially available antibodies

• Androgen stimulation controls: Cells treated with R1881 or dihydrotestosterone, which induce robust phosphorylation at Ser213

• Tissue controls: Prostate epithelial cells from samples with known Akt activation

Negative controls:

• Phosphatase treatment control: Parallel samples treated with lambda phosphatase to remove phosphorylation

• AR-negative samples: Cell lines or tissues lacking AR expression

• Primary antibody omission: To detect non-specific binding of detection systems

• Isotype control antibody: Non-specific rabbit IgG at the same concentration

Specificity controls:

• Peptide competition: Pre-incubation of antibody with excess phospho-peptide (E-A-S(p)-G-A) should abolish specific signal

• Mutant AR expression: S213A mutant AR provides an ideal negative control as it cannot be phosphorylated at this site

• Pathway inhibition: Treatment with PI3K inhibitors (e.g., LY294002) should reduce phosphorylation

Signal validation controls:

• Total AR detection: Run in parallel to confirm AR protein presence and enable normalization

• Phospho-Akt detection: Since Akt is implicated in Ser213 phosphorylation, confirm its activation status

• Cross-methodology validation: When possible, confirm findings with complementary techniques

Cell type controls:

• Mixed cell populations: Compare prostate epithelial cells (positive) vs. stromal cells (negative) within the same sample

• Proliferation markers: In developmental contexts, compare Ki67-positive cells (minimal phosphorylation) vs. differentiated cells (positive phosphorylation)

Table: Control matrix for different experimental applications

This comprehensive set of controls ensures reliable interpretation of results and helps troubleshoot technical issues with the antibodies across different experimental platforms.

How do experimental conditions affect detection of AR Ser213 phosphorylation?

Multiple experimental variables can significantly impact the detection of AR Ser213 phosphorylation and must be carefully controlled:

Hormone treatment conditions:

• Phosphorylation at Ser213 occurs robustly in response to R1881 and dihydrotestosterone but weakly if at all with testosterone

• Optimal androgen concentrations are typically 1-10 nM for R1881 and 10-100 nM for DHT

• Time course analysis shows that phosphorylation occurs with rapid kinetics following androgen stimulation

• AR antagonists do not induce phosphorylation at this site

Cell culture variables:

• Serum components contain hormones and growth factors that can affect baseline phosphorylation

• Charcoal-stripped serum is recommended for experiments examining hormone-dependent phosphorylation

• Cell density affects signaling pathway activation and should be standardized

• Duration of serum starvation influences baseline phosphorylation status

Sample preparation factors:

• Phosphatase inhibitor cocktails are absolutely essential during cell/tissue lysis

• Sample heating duration and temperature can affect phospho-epitope stability

• Flash freezing of tissues helps preserve phosphorylation status

• Time between tissue harvesting and fixation critically affects phospho-epitope preservation

Kinase activation conditions:

• PI3K/Akt pathway activity directly influences Ser213 phosphorylation

• LY294002 (PI3K inhibitor) treatment blocks phosphorylation at this site

• Growth factor stimulation alone (without androgen) does not induce significant Ser213 phosphorylation

• Cellular stress (e.g., oxidative stress, hypoxia) can alter kinase-phosphatase balance

Antibody performance variables:

• Different antibody lots may show variation in specificity and sensitivity

• Antibody concentration and incubation time should be optimized for each application

• Buffer composition (particularly BSA vs. milk-based blockers) affects phospho-detection

• Detection system sensitivity (chemiluminescence vs. fluorescence) influences signal detection

Table: Experimental conditions affecting AR Ser213 phosphorylation detection

Understanding and controlling these variables is essential for reproducible detection of AR Ser213 phosphorylation across different experimental platforms and biological systems.

How does AR Ser213 phosphorylation contribute to treatment resistance in prostate cancer?

Emerging evidence suggests that AR Ser213 phosphorylation may play a significant role in the development of treatment resistance in prostate cancer through multiple mechanisms:

Clinical evidence of association:
When phospho-Akt and phospho-AR Ser213 were assessed by immunohistochemistry in matched hormone-naïve and castration-resistant prostate cancer tumors, increases in both were observed in resistant tumors . This correlation suggests potential involvement in resistance mechanisms, though causality requires further investigation.

Interaction with AKT signaling:
The synergy between AKT and AR signaling can transform naïve prostatic epithelium into androgen-insensitive carcinoma . While phosphorylation of AR at Ser213 and Ser791 by AKT is not critical for this synergy , the increased phosphorylation observed in resistant tumors suggests it may serve as a biomarker or have alternative functions in the resistance process.

Potential molecular mechanisms:

• Phosphorylation at Ser213 may alter AR cofactor recruitment, potentially shifting from transcriptional repressors to activators in resistant cells

• Modified AR transcriptional activity may drive expression of genes promoting survival under androgen-depleted conditions

• Altered AR stability or subcellular localization influenced by this phosphorylation may contribute to persistent AR signaling

• Cross-talk with other signaling pathways may be facilitated by this phosphorylation site

Pathway interactions beyond direct phosphorylation:
While direct phosphorylation at Ser213 is not essential for the AKT-AR synergy in promoting androgen-insensitive growth , AKT may regulate AR activity through multiple mechanisms:

• Modulation of AR expression levels

• Effects on AR stability and degradation

• Influence on AR subcellular localization

• Phosphorylation of AR cofactors

Therapeutic implications:
The correlation between increased phospho-Akt and phospho-AR Ser213 in resistant tumors suggests potential therapeutic strategies:

• Combined inhibition of AR and PI3K/Akt signaling

• Development of drugs targeting AR specifically in its phosphorylated state

• Use of phospho-AR (Ser213) as a biomarker for selecting patients for specific treatment approaches

Understanding the precise role of AR Ser213 phosphorylation in treatment resistance represents an important research direction with potential clinical implications for managing advanced prostate cancer.

What is the relationship between AR Ser213 phosphorylation and other post-translational modifications?

AR Ser213 phosphorylation exists within a complex network of post-translational modifications (PTMs) that collectively regulate AR function:

Relationship with other AR phosphorylation sites:

The human AR contains multiple phosphorylation sites including Ser-16, Ser-81, Ser-94, Ser-256, Ser-308, Ser-424, Ser-515, Ser-650, and Ser-791 . These sites form an interconnected regulatory network:

Hierarchical phosphorylation patterns:

Evidence suggests that AR phosphorylation follows hierarchical patterns, with some sites serving as "priming" sites for subsequent phosphorylation events:

• After protein synthesis, Ser-650 is phosphorylated first, followed by Ser-94 in the second isoform

• Hormone stimulation leads to increased phosphorylation across multiple sites

• The S515A mutation affects phosphorylation at distant sites, suggesting long-range structural effects

Interplay with other post-translational modifications:

AR phosphorylation interacts with other types of post-translational modifications:

Conformational effects:

Phosphorylation at Ser213 may induce conformational changes affecting:

• AR N/C terminal interaction

• DNA binding capacity

• Cofactor binding surfaces

• Nuclear localization sequence accessibility

Kinase-specific effects:

Different kinases can target the same sites under different conditions:

Understanding this complex interplay between AR Ser213 phosphorylation and other post-translational modifications represents an important frontier in AR biology research with implications for therapeutic targeting.