Phospho-CBX5 (S92) Antibody
Description
Introduction to Phospho-CBX5 (S92) Antibody
The Phospho-CBX5 (S92) antibody is a specialized immunochemical tool designed to detect Chromobox Protein Homolog 5 (CBX5, also known as HP1α) when phosphorylated at serine 92. This post-translational modification is critical for regulating HP1α’s role in mitotic chromosome dynamics and genomic stability . Unlike standard CBX5 antibodies, this phosphospecific variant exclusively binds the activated (phosphorylated) form of the protein, enabling precise tracking of its functional state in cellular processes .
Biological Context and Target Protein
CBX5/HP1α is a chromatin-associated protein involved in heterochromatin formation, gene silencing, and mitotic regulation. Phosphorylation at serine 92 (S92) by Aurora Kinase B (AURKB) occurs during early prophase and is essential for:
Research Findings and Functional Insights
Key discoveries from studies using Phospho-CBX5 (S92) antibodies include:
Mitotic Regulation
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Temporal Specificity: S92 phosphorylation peaks during mitosis (8–10 hours post-synchronization), coinciding with AURKB and Cyclin B1 expression .
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Subcellular Localization: Phosphorylated HP1α colocalizes with AURKB and CENPA at centromere-kinetochore domains during prometaphase (Figure 1) .
Chromosomal Stability
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The phosphomimetic S92D mutant reduced chromosomal abnormalities by 38–51%, while the non-phosphorylatable S92A mutant exacerbated defects .
Protein Interactions
Phosphorylated HP1α associates with:
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INCENP (chromosome passenger complex).
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CENPA (centromere-specific histone variant).
Experimental Applications and Protocols
Phospho-CBX5 (S92) antibodies are validated for:
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Immunofluorescence (IF): Detects centromeric HP1α in mitotic cells using protocols involving EDTA-based antigen retrieval and DyLight®488-conjugated secondary antibodies .
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Western Blot (WB): Identifies phosphorylated HP1α (~25 kDa band) in synchronized cell lysates .
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Immunohistochemistry (IHC): Used in paraffin-embedded cancer tissues (e.g., breast, ovarian, rectal carcinomas) with Strepavidin-Biotin-Complex (SABC) and DAB visualization .
Implications in Disease and Therapeutics
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Cancer: Dysregulated S92 phosphorylation correlates with chromosomal instability, a hallmark of malignancies like renal clear cell carcinoma and ovarian serous adenocarcinoma .
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Therapeutic Targeting: AURKB inhibitors (e.g., Barasertib) indirectly modulate HP1α phosphorylation, offering avenues for mitigating mitotic errors in cancer .
Table 1: Phospho-CBX5 (S92) Antibody Performance
| Application | Sample Type | Dilution | Detection Method |
|---|---|---|---|
| IF/ICC | A549 cells | 5 μg/mL | DyLight®488, DAPI |
| IHC | Paraffin-embedded | 2 μg/mL | SABC, DAB |
| WB | HeLa cell lysates | 1:1,000 | Chemiluminescence |
Table 2: Key Phosphorylation Sites in HP1α
| Residue | Kinase | Functional Role |
|---|---|---|
| S92 | Aurora Kinase B | Centromere localization |
| S177 | Unknown | Heterochromatin maintenance |
Product Specs
Target Background
- Our research has focused on interactions between HP1alpha and the chromosomal passenger complex (CPC) during mitosis and interphase. Our studies using artificial tethering of HP1alpha to centromeres revealed a robust interaction between this protein and the CPC. PMID: 29467217
- These data suggest that upregulated HP1alpha, SUV39H1, and H3K9me3 in glioma cells are functionally linked to glioma pathogenesis and progression, and could potentially serve as novel biomarkers for future diagnostic and therapeutic targeting of brain tumors. PMID: 28946550
- Data suggest that SUMOylated HP1alpha is a critical epigenetic regulator of DNA repair in breast cancer (BCa), potentially determining chemotherapy responsiveness. PMID: 27107417
- Our findings suggest that heterochromatin-mediated gene silencing may partially occur through sequestration of compacted chromatin within phase-separated HP1 droplets, which are dissolved or formed by specific ligands depending on the nuclear context. PMID: 28636604
- Data show that SALL4 promotes the expression of Glut1 and open chromatin through a HP1alpha-dependent mechanism. PMID: 28759035
- These data suggest that upregulated HP1A and H3K9me3 in glioma cells are functionally associated with glioma pathogenesis and progression. PMID: 28623138
- Loss of HP1alpha and gamma isoforms inhibits the upregulation of Suv39h1 and H3K9me3 observed under stress conditions. PMID: 28059589
- We demonstrated the essential role of HP1 in regulating HR through BRCA1/BARD1-mediated accumulation of FANCJ and CtIP at DSB sites. This mechanism may affect tumorigenesis and chemosensitivity, making it of high clinical significance. PMID: 27399284
- The dynamic string-like behavior of HP1alpha's N-terminal tail underlies the enhancement in H3 binding due to phosphorylation. PMID: 26934956
- We demonstrate that an hnRNPA1 and CBX5 bi-directional core promoter fragment does not possess intrinsic capacity for specific CBX5 down-regulation in metastatic cells. PMID: 26791953
- HP1alpha plays an important role in the differentiation and angiogenic function of Endothelial Progenitor Cells. PMID: 25588582
- Jra recruits the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation. PMID: 25945750
- HP1 regulates this gene's alternative splicing in a methylation-dependent manner by recruiting splicing factors to its methylated form. PMID: 25704815
- PIP5K1A modulates ribosomal RNA gene silencing through its interaction with histone H3 lysine 9 trimethylation and heterochromatin protein HP1-alpha. PMID: 26157143
- Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications. PMID: 25519718
- The results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes. PMID: 25332400
- Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin, and plays a crucial role in forming higher-order chromatin structures. PMID: 24825911
- CRL4B promotes tumorigenesis by coordinating with SUV39H1/HP1/DNMT3A in DNA methylation-based epigenetic silencing. PMID: 24292684
- RAD6 physically interacts with HP1alpha and ubiquitinates HP1alpha at residue K154, promoting HP1alpha degradation through the autophagy pathway. PMID: 25384975
- These findings reveal a previously unrecognized but direct link between HP1 and CPC localization in the centromere, highlighting the critical role of borealin-HP1 interaction in orchestrating accurate cell division. PMID: 24917673
- Spatiotemporal dynamics of HP1A localization to centromere is governed by two distinct structural determinants. PMID: 25104354
- The phosphorylation-dephosphorylation cycle of HP1alpha orchestrates accurate progression of cells through mitosis. PMID: 24786771
- Binding of HP1alpha, HP1beta, and HP1gamma to the globular domain of histone H3 is differentially regulated by phosphorylation of residues H3T45 and H3S57. PMID: 24820035
- CTCF may regulate vigilin behavior, indirectly influencing the binding of HP1alpha to the satellite 2 locus. PMID: 24561205
- These findings demonstrate that HP1(Hsalpha)-nucleosome interactions cause chromatin condensation, a process that regulates numerous chromosome events. PMID: 24415761
- HP1alpha mutation W174A, which disrupts interactions with proteins containing the PxVxL motif, did not affect interactions with the BZip protein. The HP1alpha W41A mutation, which prevents binding to methylated histones, exhibited greatly reduced FRET efficiency. PMID: 23392382
- HP-1alpha/beta may be useful in the differential diagnosis of renal tumors, particularly in differentiating chromophobe RCC and oncocytoma. PMID: 23142018
- We have identified CBX5 as a potential target regulating lung cancer survival and the stem-like properties of lung CD133+- tumor stem-like cells (TSLCs). PMID: 22900142
- The hinge region (HR) connecting the CD and C-terminal chromoshadow domain (CSD), and the CSD contributed to the selective binding of HP1alpha to histone H3 with trimethylated lysine 9 through weak DNA binding and by suppressing the DNA binding, respectively. PMID: 23142645
- HP1-alpha and PADI4 are regulators of both immune genes and HERVs, suggesting that multiple events of transcriptional reactivation in Multiple Sclerosis patients could be explained by the deficiency of a single mechanism of gene silencing. PMID: 23028349
- Downregulation of the telomeric noncoding RNA requires SUV39H1 and HP1A. PMID: 22922742
- These studies reveal a novel role for HP1 as a cofactor in tumor suppression, expanding our mechanistic understanding of a KLF associated to human disease. PMID: 22318730
- HP1 increases the chromatin association of VHL. PMID: 22234250
- The finding that HP1 alpha is down-regulated primarily at the transcriptional level provides new insight for further elucidation of the detailed molecular mechanisms causing the HP1 alpha down-regulation in invasive breast cancer cells. PMID: 21374739
- A link between mutant codanin-1 and the aberrant localization of HP1 alpha is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1 alpha antibodies in erythroblasts from patients with congenital dyserythropoietic anemia type 1. PMID: 21364188
- HP1alpha binding by INCENP or Shugoshin 1 (Sgo1) is dispensable for centromeric cohesion protection during mitosis of human cells, but might regulate yet unknown interphase functions of the chromosome passenger complex (CPC) or Sgo1 at the centromeres. PMID: 21346195
- Recent findings and controversies concerning HP1 functions in mammalian cells, in comparison to studies in other organisms, are reviewed. PMID: 20421743
- ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. PMID: 20154359
- The assembly of HP1 in the inner centromere and the localization of hMis14 at the kinetochore are mutually dependent in human chromosomes. PMID: 20231385
- Data suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin, and that HP1 proteins utilize this property to detect and arrest unwanted chromatin remodeling. PMID: 20011120
- HP1alpha expression regulation is dependent on cell proliferation. PMID: 20049717
- Heterochromatin protein 1 is extensively decorated with histone code-like post-translational modifications. PMID: 19567367
- Reduction of YY1 expression in breast cancer cells could contribute to the acquisition of an invasive phenotype through increased cell migration as well as by reduced expression of HP1alpha. PMID: 19566924
- These results suggested that, although the majority of HP1alpha diffuses into the cytoplasm, some populations are retained in the centromeric region and involved in the association and segregation of sister kinetochores during mitosis. PMID: 11942629
- Identification of three amino acid residues I113, A114 and C133 in HP1alpha that are essential for the selective interaction of HP1alpha with BRG1. PMID: 12411497
- Developmentally regulated ARL5, with its distinctive nuclear/nucleolar localization and interaction with HP1alpha, may play a role(s) in nuclear dynamics and/or signaling cascades during embryonic development. PMID: 12414990
- Histone H3 methylase Suv39h1 and the methyl lysine-binding protein HP1alpha directly interact with MBD of MBD1 in vitro and in cells. PMID: 12711603
- HP1 has a role in the recruitment but not in the stable association of Orc1p with heterochromatin. PMID: 15454574
- HP1alpha recruits endogenous HP1beta to the chromatin, inducing heterochromatin formation and enhanced histone lysine methylation. PMID: 15899859
- Results describe the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells, and show its colocalization with heterochromatin protein 1 orthologs in pericentric heterochromatin. PMID: 16487625
Q&A
What is Phospho-CBX5 (S92) antibody and what does it specifically detect?
The Phospho-CBX5 (S92) antibody specifically detects endogenous levels of Heterochromatin Protein 1 alpha (HP1α, also known as CBX5) only when phosphorylated at serine 92 . This specificity makes it a valuable tool for studying the phosphorylated state of HP1α in various cellular processes. The antibody has been developed as a rabbit polyclonal antibody raised against synthetic phosphopeptide derived from human HP1α around the phosphorylation site of Ser92 .
What is the biological significance of CBX5/HP1α phosphorylation at serine 92?
Phosphorylation of HP1α at serine 92 plays a critical role in chromosomal stability during cell division. Research indicates that S92-phosphorylated HP1α is generated in cells at early prophase, localizes to centromeres, and associates with regulators of chromosome stability . This phosphorylation appears to be involved in the proper formation of kinetochores through interaction with essential kinetochore proteins . The phosphorylation state of HP1α at S92 thus represents a key regulatory mechanism in mitotic progression and chromosome segregation.
What is the cellular localization pattern of phosphorylated HP1α?
Phosphorylated HP1α (P-S92-HP1α) exhibits specific localization patterns depending on the cell cycle stage. It colocalizes with Aurora B kinase (AURKB) and INCENP at centromere-kinetochore domains during early mitosis . There is also a non-chromatin-associated cytoplasmic pool of phosphorylated HP1α that appears to be regulated independently of AURKB, suggesting multiple kinases may be involved in HP1α phosphorylation at different cellular locations .
What are the recommended applications for Phospho-CBX5 (S92) antibody?
The Phospho-CBX5 (S92) antibody has been validated for several research applications:
Researchers should optimize dilution for their specific experimental conditions and sample types .
How should samples be prepared for optimal detection of phosphorylated CBX5?
For optimal detection of phosphorylated CBX5, careful sample preparation is essential. When performing immunofluorescence studies, cells should be fixed with paraformaldehyde and permeabilized appropriately to preserve phospho-epitopes. For detection of phosphorylated HP1α during mitosis, synchronization of cells with nocodazole may be beneficial . When performing western blots, phosphatase inhibitors must be included in lysis buffers to prevent dephosphorylation of the target protein. For immunohistochemistry, proper antigen retrieval is critical, with TE buffer pH 9.0 generally recommended for phospho-epitopes .
What are the optimal storage conditions for maintaining antibody activity?
For maximum stability and performance, Phospho-CBX5 (S92) antibody should be stored at -20°C or -80°C . The antibody is typically provided in a storage buffer containing PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide . Repeated freeze-thaw cycles should be avoided as they can degrade antibody performance . For longer-term storage, aliquoting the antibody into smaller volumes is recommended to minimize freeze-thaw cycles .
What role does phosphorylated HP1α play in chromosomal stability?
Phosphorylated HP1α at S92 plays a critical role in maintaining chromosomal stability during cell division. Experimental evidence from MEF (Mouse Embryonic Fibroblast) models with Cbx5 deletion showed increased chromosomal instability, manifested as anaphase bridges and micronuclei . Rescue experiments using adenovirus-mediated delivery of wild-type or mutant HP1α revealed that the phosphomimetic (S92D) mutant significantly reduced both chromatin bridges and micronuclei compared to control cells . This suggests that phosphorylation of HP1α at S92 is important for preventing chromosomal abnormalities during mitosis, potentially through its role in kinetochore formation and centromere function .
How does the phosphorylation state of HP1α affect its interaction with other chromatin-associated proteins?
The phosphorylation of HP1α at S92 appears to modulate its interactions with other chromatin-associated proteins, particularly those involved in kinetochore assembly and function. Proximity ligation assays (PLA) have been used to detect specific protein-protein interactions involving P-S92-HP1α . While HP1α normally recognizes and binds histone H3 tails methylated at 'Lys-9' (H3K9me) leading to epigenetic repression, its phosphorylation may alter this interaction or enable additional protein-protein interactions . The phosphorylation state likely serves as a molecular switch that changes HP1α's binding partners during different stages of the cell cycle, particularly during mitosis when it localizes to centromeres .
What are appropriate controls when using Phospho-CBX5 (S92) antibody?
When using the Phospho-CBX5 (S92) antibody, several controls should be incorporated to ensure valid results:
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Positive control: Use cell lines or tissues known to express phosphorylated HP1α, such as HeLa cells during mitosis .
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Negative controls:
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Specificity controls:
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Knockdown/knockout validation: Use CBX5 knockdown or knockout cells (like Cbx5-/- MEFs) to confirm antibody specificity .
How can inconsistent staining patterns with Phospho-CBX5 (S92) antibody be resolved?
Inconsistent staining patterns when using Phospho-CBX5 (S92) antibody may be resolved through the following approaches:
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Optimize fixation conditions: Phospho-epitopes can be sensitive to overfixation. Test different fixation times and methods to preserve the phosphorylation state.
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Include phosphatase inhibitors: Always use fresh phosphatase inhibitors in lysis buffers and during sample preparation to prevent dephosphorylation.
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Optimize antibody concentration: Titrate the antibody using a range of dilutions (e.g., 1:50, 1:100, 1:200, 1:300) to determine optimal signal-to-noise ratio .
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Adjust antigen retrieval method: For IHC applications, try both TE buffer pH 9.0 and citrate buffer pH 6.0 to determine which better exposes the phospho-epitope .
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Consider cell cycle stage: Since P-S92-HP1α levels vary throughout the cell cycle, synchronize cells or analyze cell cycle stage to ensure consistent results .
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Prevent antibody degradation: Aliquot antibody to avoid repeated freeze-thaw cycles and store according to manufacturer recommendations .
How can researchers distinguish between specific and non-specific signals when using Phospho-CBX5 (S92) antibody?
Distinguishing between specific and non-specific signals requires careful experimental design:
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Compare with known localization patterns: Specific P-S92-HP1α signals should show centromeric localization during early prophase and throughout mitosis .
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Use phosphatase treatment: Treat parallel samples with lambda phosphatase to confirm that the signal depends on phosphorylation status.
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Employ phosphomimetic and non-phosphorylatable mutants: Compare staining patterns in cells expressing wild-type HP1α, S92A (non-phosphorylatable), and S92D (phosphomimetic) HP1α .
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Perform dual staining: Co-stain with markers of centromeres/kinetochores (e.g., AURKB, INCENP) to confirm the expected colocalization pattern .
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Use cross-validation: Confirm results using multiple detection methods (e.g., IF, WB, IP) to increase confidence in specificity.
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Quantify colocalization: Use appropriate software to calculate coefficients such as Manders overlap coefficient (MOC) to quantitatively assess colocalization with expected partners .
How does the phosphorylation of HP1α at S92 differ functionally from other post-translational modifications of HP1α?
HP1α is subject to multiple post-translational modifications including phosphorylation at different residues, methylation, acetylation, and SUMOylation. The S92 phosphorylation appears to be specifically involved in mitotic functions and chromosomal stability . While HP1α generally recognizes and binds histone H3 tails methylated at 'Lys-9' (H3K9me) to promote heterochromatin formation and gene silencing , its phosphorylation at S92 seems to confer specialized functions during mitosis by altering its localization and interaction partners . Unlike some other modifications that may affect HP1α's role in heterochromatin formation throughout interphase, S92 phosphorylation appears to be a mitosis-specific regulatory mechanism .
What methodological approaches can be used to study the dynamics of HP1α phosphorylation in live cells?
Studying the dynamics of HP1α phosphorylation in live cells requires specialized approaches:
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Fluorescent protein fusions with phospho-sensors: Generate constructs with HP1α fused to phosphorylation-sensitive fluorescent reporters.
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FRET-based sensors: Design FRET pairs that change conformation upon HP1α phosphorylation.
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Live-cell compatible phospho-specific antibody fragments: Use Fab fragments of the Phospho-CBX5 (S92) antibody conjugated to cell-permeable peptides.
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Optogenetic approaches: Use light-controllable kinases to induce HP1α phosphorylation at specific times and locations.
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Complementary fixed-cell time course analysis: Perform detailed immunofluorescence using Phospho-CBX5 (S92) antibody on cells fixed at precisely timed intervals throughout mitosis .
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Correlative live-cell/fixed-cell microscopy: Track cells expressing fluorescently tagged HP1α in real-time, followed by fixation and staining with Phospho-CBX5 (S92) antibody at specific points.
