Phospho-AR (S213) Antibody
Description
Role in AR Phosphorylation Studies
Phosphorylation at S213 is mediated by kinases such as Akt and PIM1, influencing AR stability, nuclear translocation, and transcriptional activity . The antibody has been instrumental in:
-
Detecting PIM1-mediated phosphorylation: Overexpression of PIM1 in LNCaP cells increases S213 phosphorylation, which correlates with altered AR target gene expression (e.g., PSA) .
-
Investigating cross-talk with signaling pathways: S213 phosphorylation is PI3K/Akt-dependent and linked to ligand-independent AR activation via CXCL12/CXCR4 signaling .
Clinical Relevance in Prostate Cancer
-
Castration-resistant prostate cancer (CRPC): S213 phosphorylation is associated with higher-grade tumors and resistance to androgen deprivation therapy. Antibody-based studies confirm elevated phosphorylation in CRPC models .
-
Prognostic biomarker potential: Immunohistochemical detection of phosphorylated AR at S213 in primary tumors correlates with aggressive disease and therapeutic resistance .
Mechanistic Insights
-
Protein-protein interactions: The antibody has shown that S213 phosphorylation facilitates binding of the coactivator 14-3-3 ζ, enhancing AR transcriptional activity in low-androgen conditions .
-
Degradation pathways: PIM1S isoform-mediated S213 phosphorylation recruits Mdm2, an E3 ligase, promoting AR ubiquitination and degradation .
Antibody Validation Data
Optimized Protocols
Product Specs
Target Background
- AR expression heterogeneity is linked to distinct castration/enzalutamide responses in castration-resistant prostate cancer. PMID: 30190514
- Androgen receptor positive triple negative breast cancer: Clinicopathologic, prognostic, and predictive features PMID: 29883487
- In prostate cancer cells, AR-V7 expression is correlated with drug resistance. Upregulation of AR-V7 enhances proliferation potency of cancer cells, indicating unfavorable prognosis for patients. PMID: 30284554
- These findings suggest that a deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype androgen receptor mRNA, leading to partial androgen insensitivity syndrome. PMID: 29396419
- Germline Mutations and Polymorphisms in AR were associated with Prostate Cancer. PMID: 30139231
- GTEE also downregulated the expression of AR and prostate-specific antigen (PSA) in both androgen-responsive and castration-resistant PCa cells. By blocking the SREBP-1/AR axis, GTEE suppressed cell growth and progressive behaviors, and activated the caspase-dependent apoptotic pathway in PCa cells. PMID: 30301150
- Suppressed the expression of androgen receptor. PMID: 29981500
- An AR motif of the transactivation domain has been identified that contributes to transcriptional activity by recruiting the C-terminal domain of subunit 1 of the general transcription regulator TFIIF. PMID: 29225078
- In LNCaP prostate cancer cells, TSG101 overexpression recruits the androgen receptor (AR) to TSG101-containing cytoplasmic vesicles, leading to reduced AR protein level and downregulation of AR transactivation activity. Immunofluorescence microscopy demonstrated that TSG101-decorated cytoplasmic vesicles are associated with late endosomes/lysosomes. PMID: 29859188
- Study indicates that both mRNA and protein level of AR increase during prostate cancer (PCa) progression. These levels are even higher in metastatic PCa. Further data suggest that elevation of AR may promote PCa metastasis by induction of EMT and reduction of KAT5. PMID: 30142696
- This study aimed to determine the presence and localization of oestrogen receptors (ERs), progesterone receptors (PRs), and androgen receptors (ARs) in both healthy and varicose vein wall cells and their relationship with gender. PMID: 30250632
- These findings suggest that CDK11 is involved in the regulation of AR pathway and AR can be a potential novel prognostic marker and therapeutic target for osteosarcoma treatment. PMID: 28262798
- We use the CPRC prostate cancer model and demonstrate that endothelial cells secrete large amounts of CCL5 and induce autophagy by suppressing AR expression in prostate cancer cell lines. Consequently, elevated autophagy accelerates focal adhesions proteins disassembly and promotes prostate cancer invasion. Inhibition of both CCL5/CCR5 signaling and autophagy significantly reduces metastasis in vivo. PMID: 30200999
- Overexpression of nuclear AR-V7 protein identifies a subset of tumors with remarkably aggressive growth characteristics among clinically and histologically high-risk patients at the time of radical prostatectomy. PMID: 29198908
- Study defines AR ligand-binding domain homodimerization as an essential step in the proper functioning of this important transcription factor. The dimerization surface harbors over 40 previously unexplained androgen insensitivity syndromes and prostate cancer-associated point mutations. PMID: 28165461
- Loss of AR expression was found in the nucleus of penile cancer cells when compared to normal tissues. Cytoplasmic AR immunostaining was observed in a significant number of these cases and was related with poor prognosis and shorter overall survival. PMID: 30099587
- The AR polymorphism is associated with POR risk, patients with repeats greater than 22 show a higher risk. Our data suggest that AR genotype could play a role in natural ovarian aging. PMID: 29886316
- In all, these data suggest that Aurora A plays a pivotal role in regulation of Androgen receptor variant 7 expression and represents a new therapeutic target in castrate-resistant prostate cancer. PMID: 28205582
- The meta-analysis showed that short CAG and GGN repeats in androgen receptor gene were associated with increased risk of prostate cancer, especially in Caucasians. PMID: 28091563
- Knockdown of beta-Klotho produced the opposite effects. In conclusion, beta-Klotho inhibits EMT and plays a tumorsuppressive role in prostate cancer (PCa) , linking FGF/FGFR/beta-Klotho signaling to the regulation of PCa progression. PMID: 29749458
- The interaction of AR and SP1 contributes to regulate EPHA3 expression. PMID: 29917167
- DHX15 regulates androgen receptor (AR) activity by modulating E3 ligase Siah2-mediated AR ubiquitination independent of its ATPase activity promoting prostate cancer progression. PMID: 28991234
- The interaction of Nanog with the AR signaling axis might induce or contribute to Ovarian cancer stem cells regulation. In addition, androgen might promote stemness characteristics in ovarian cancer cells by activating the Nanog promoter. PMID: 29716628
- A significant subset of endometrial cancers express androgen receptor, especially serous cancers. PMID: 29747687
- Letter: eradication of androgen receptor amplification, PSA decline, and clinical improvement with high dose testosterone therapy. PMID: 28040353
- The results in this meta-analysis indicated that AR CAG and GGN repeat polymorphisms may be an important pathogenesis of cryptorchidism. PMID: 29044734
- The inverse relation observed between bone cell activity and tumor cell AR activity in prostate cancer bone metastasis may be of importance for patient response to AR. PMID: 29670000
- Length variations of (CAG)n and (GGC)n polymorphism in the transactivation domain of AR, significantly influence hormonal profile, semen parameters, and sexual functions of asthenospermic subjects by down regulating the expression of AR mediating signaling. PMID: 29083935
- Data suggest that somatic mosaicism in AR can cause partial androgen insensitivity syndrome. [CASE REPORT] PMID: 29267169
- These results identify HoxB13 as a pivotal upstream regulator of AR-V7-driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7-driven prostate tumors. PMID: 29844167
- TRX1 is an actionable castration-resistant prostate cancer therapeutic target through its protection against AR-induced redox stress. PMID: 29089489
- These findings reveal AR-genomic structural rearrangements as important drivers of persistent AR signalling in castration-resistant prostate cancer. PMID: 27897170
- AR+ was associated with lower breast cancer mortality in the overall study population ( estrogen receptor-negative). PMID: 28643022
- Nuclear COBLL1 interacts with AR to enhance complex formation with CDK1 and facilitates AR phosphorylation for genomic binding in castration-resistant prostate cancer model cells. PMID: 29686105
- A variety of AR mutants are induced under selective pressures of AR pathway inhibition in castration resistant prostate cancer which remain sensitive to the inhibitor darolutamide. PMID: 28851578
- c.3864T>C AR novel mutation is responsible for complete androgen insensitivity syndrome [case report] PMID: 29206494
- The Spinal and bulbar muscular atrophy is caused by the expansion of a CAG/glutamine tract in the amino-terminus of the androgen receptor PMID: 29478604
- Polysomic AR genes show low methylation levels and high AR protein expression on immunohistochemistry PMID: 29802469
- Oral administration of RAD140 substantially inhibited the growth of AR/ER(+) breast cancer patient-derived xenografts (PDX). Activation of AR and suppression of ER pathway, including the ESR1 gene, were seen with RAD140 treatment. PMID: 28974548
- The aims of this study was to evaluate if extreme CAG and GGN repeat polymorphisms of the androgen receptors influence body fat mass, its regional distribution, resting metabolic rate, maximal fat oxidation capacity and serum leptin, free testosterone and osteocalcin in healthy adult men PMID: 29130706
- The CRISPR/Cas9 system was able to edit the expression of AR and restrain the growth of androgen-dependent prostate cancer cells in vitro, suggesting the potential of the CRISPR/Cas9 system in future cancer therapy. PMID: 29257308
- A new mechanism for complete androgen insensitivity syndrome (CAIS). A deep intronic pseudoexon-activating mutation in the intron between exons 6 and 7 of AR, detected in two siblings with CAIS, leads to aberrant splicing of the AR mRNA and insufficient AR protein production. PMID: 27609317
- In the current work, we have confirmed that the lead androgen receptor DBD inhibitor indeed directly interacts with the androgen receptor DBD and tested that substance across multiple clinically relevant castration-resistant prostate cancer cell lines. PMID: 28775145
- Androgen receptor CAG repeat polymorphism is not associated with insulin resistance and type 2 diabetes in Sri Lankan males. PMID: 29202793
- AR gene CAG repeat polymorphisms are associated with the increased risk of mild endometriosis PMID: 28915409
- ARE full sites generate a reliable transcriptional outcome in AR positive cells, despite their low genome-wide abundance. In contrast, the transcriptional influence of ARE half sites can be modulated by cooperating factors. PMID: 27623747
- Targeting the Malat1/AR-v7 axis via Malat1-siRNA or ASC-J9 can be developed as a new therapy to better suppress enzalutamide-resistant prostate cancer progression. PMID: 28528814
- High circulating AR-V7 levels predicted resistance to abiraterone and enzalutamide in castration-resistant prostate cancer. PMID: 28818355
- Results identified the N-terminal region of AR-V7 (splice variants) that interacts with the diffuse B-cell lymphoma homology (DH) domain of Vav3 which increases its expression in castration-resistant prostate cancer (CRPC). PMID: 28811363
- The single nucleotide polymorphism G1733A of the androgen receptor gene is significantly associated with recurrent spontaneous abortions in Mexican patients. PMID: 28707146
Q&A
What experimental applications are validated for Phospho-AR (S213) antibodies?
Phospho-AR (S213) antibodies are primarily validated for western blot (WB) and immunocytochemistry/immunofluorescence (ICC/IF) . Key methodological considerations include:
-
Sample preparation: Methanol fixation preserves epitope integrity for ICC/IF, while RIPA buffer extraction is optimal for WB .
-
Specificity controls: Co-incubation with immunizing peptide (e.g., E-A-S(p)-G-A) abolishes signal, confirming antibody specificity . Lambda phosphatase treatment should eliminate phosphorylation-dependent signals, as demonstrated in 293 and LNCaP cell models .
-
Cross-reactivity: No detectable binding to non-phosphorylated AR or phosphorylation sites outside S213, as shown by lack of signal in S213A mutant AR assays .
Which cell lines and tissue models are appropriate for studying AR-S213 phosphorylation?
-
Androgen-sensitive models: LNCaP and VCaP cells show ligand-dependent AR-S213 phosphorylation, detectable via immunoblotting after R1881 treatment .
-
Androgen-resistant models: LNCaP-abl (abl) sublines exhibit constitutive phosphorylation even in hormone-depleted conditions, mimicking clinical castration resistance .
-
Clinical specimens: Prostate cancer tissue microarrays (TMAs) with hormone-refractory samples show robust P-AR S213 staining, correlating with Gleason score ≥7 and biochemical recurrence .
How do researchers distinguish PIM1-mediated phosphorylation from Akt-mediated AR-S213 modification?
While both kinases target S213, methodological differentiation involves:
-
Kinase inhibition: PIM1/2 inhibitor V reduces phosphorylation in a dose-dependent manner, whereas Akt inhibitors (e.g., MK2206) show minimal effect .
-
In vitro kinase assays: Recombinant GST-PIM1 phosphorylates AR-S213 independent of ligand, whereas His-Akt requires co-activators (e.g., PIP3) and fails to phosphorylate AR in vitro .
-
Cellular localization: PIM1’s nuclear isoform (33 kDa) colocalizes with AR in hormone-treated cells, whereas Akt-driven phosphorylation occurs primarily in cytoplasmic compartments .
How do contradictory findings about AR-S213 phosphorylation’s functional outcomes arise, and how can they be resolved?
Conflicting reports on AR-S213’s role in transcriptional activation stem from:
-
Cellular context: In LNCaP cells, PIM1 overexpression suppresses PSA expression but upregulates IL6, suggesting promoter-specific effects .
-
Kinase crosstalk: Akt upregulates total AR protein levels, indirectly amplifying phosphorylation signals without directly modifying S213 .
Resolution strategies:
-
Use S213A mutant AR to isolate phosphorylation-specific effects .
-
Combine kinase-specific inhibitors with siRNA knockdown to delineate PIM1 vs. Akt contributions.
What advanced techniques are required to map AR-S213 phosphorylation dynamics in vivo?
-
Proximity ligation assays (PLA): Quantify kinase-AR interactions in intact tissues using PIM1/AR or Akt/AR antibody pairs.
-
Phos-tag SDS-PAGE: Resolve phosphorylation stoichiometry across clinical samples, as demonstrated in TMAs .
-
CUT&RUN sequencing: Profile genome-wide AR-S213 binding sites in hormone-refractory vs. naive tumors.
How does AR-S213 phosphorylation influence therapeutic resistance mechanisms?
Mechanistic insights:
-
Ligand-independent activation: Phosphorylated AR recruits MED1/DRIP205 coactivators in the absence of androgens, sustaining proliferation in abl cells .
-
Immune evasion: Upregulated IL6 (via phospho-AR) induces PD-L1 expression in tumor-associated fibroblasts, as observed in co-culture models .
Therapeutic implications:
How can researchers validate antibody specificity for AR-S213 in multiplex kinase environments?
-
Step 1: Validate via kinase-addback experiments in AR-null cells (e.g., DU145) transfected with WT AR vs. S213A mutant .
-
Step 2: Perform peptide competition assays using phosphorylated vs. non-phosphorylated S213 peptides (10:1 molar ratio) .
-
Step 3: Utilize mass spectrometry to confirm phosphorylation occupancy at S213 in immunoprecipitated AR .
What are the limitations of current Phospho-AR (S213) antibodies in clinical biomarker studies?
-
Heterogeneity: Polyclonal antibodies (e.g., ab131346) may exhibit batch-to-batch variability, necessitating parallel testing with monoclonal reagents .
-
Fixation artifacts: Prolonged formalin fixation masks epitopes, requiring antigen retrieval optimization (citrate buffer, pH 6.0, 95°C, 20 min) .
How can single-cell technologies refine our understanding of AR-S213’s role in tumor heterogeneity?
-
scRNA-seq: Cluster tumor subpopulations based on AR phosphorylation status and PIM1/Akt expression.
-
CyTOF: Quantify phospho-AR, PIM1, and immune checkpoint markers (PD-1/PD-L1) in circulating tumor cells.
