Phospho-CTBP1 (S422) Antibody

What is CTBP1 and what function does phosphorylation at S422 serve?

CTBP1 (C-terminal binding protein 1) is a transcriptional co-repressor that binds to the C-terminus of adenovirus E1A proteins. It functions as a metabolic sensor that links changes in cellular metabolism to transcriptional regulation. Phosphorylation at S422 is particularly significant as it regulates protein stability. When phosphorylated at S422 by HIPK2, CTBP1 undergoes proteasomal degradation, thereby affecting its co-repressor functions . This post-translational modification represents a critical regulatory mechanism that influences CTBP1's involvement in cellular proliferation, differentiation, and potentially oncogenic activities.

How does CTBP1 phosphorylation at S422 differ from other phosphorylation sites?

CTBP1 undergoes phosphorylation at multiple sites, each with distinct functional consequences. While S422 phosphorylation by HIPK2 primarily induces proteasomal degradation , phosphorylation at Ser158 by p21-activated kinase (Pak1), AMPK, or other kinases facilitates cytoplasmic localization and downregulates transcriptional activity . Akt1-mediated phosphorylation at Thr176 also leads to proteasomal degradation . These site-specific phosphorylation events represent separate regulatory mechanisms that can be studied with site-specific antibodies to distinguish between different cellular signaling pathways affecting CTBP1 function.

What is the evolutionary conservation of the S422 phosphorylation site in CTBP1?

While the search results don't explicitly detail evolutionary conservation of the S422 site, they do mention conservation of other phosphorylation sites (such as the Akt1 sites) among vertebrate species . The consistent availability of S422 phospho-specific antibodies with reactivity across human, mouse, and rat species suggests conservation of this site across mammals. Researchers should consider performing sequence alignments of CTBP1 from different species to fully assess conservation when designing cross-species experiments.

What are the optimal applications for Phospho-CTBP1 (S422) antibodies, and what dilutions should be used?

Phospho-CTBP1 (S422) antibodies have been validated for multiple applications with specific recommended dilutions:

The antibody demonstrates consistent detection of endogenous levels of CTBP1 when phosphorylated at S422 across these applications . For optimal results, researchers should include appropriate controls, particularly peptide competition assays to confirm specificity, as demonstrated in the immunohistochemical staining of human brain tissue .

How can researchers validate the specificity of Phospho-CTBP1 (S422) antibodies in their experimental system?

To validate antibody specificity, researchers should employ multiple approaches:

• Peptide competition assay: Pre-incubate the antibody with the immunizing phosphopeptide before application to samples. This should abolish specific signals, as demonstrated in immunohistochemical staining of human brain tissue .

• Phosphatase treatment: Treat cell lysates or tissue samples with lambda phosphatase before immunoblotting to confirm that the signal depends on phosphorylation.

• Genetic approaches: Use CRISPR/Cas9 to generate CTBP1 knockout cells as negative controls, or specifically mutate S422 to alanine to prevent phosphorylation.

• Stimulation experiments: Treat cells with factors known to induce S422 phosphorylation (potentially TNF, as suggested by Western blot analysis of TNF-treated Jurkat cells ).

• Compare with total CTBP1 antibody signals to distinguish between changes in phosphorylation versus total protein levels.

What experimental conditions can induce or inhibit CTBP1 S422 phosphorylation?

Based on the available data, researchers can modulate CTBP1 S422 phosphorylation through several approaches:

• TNF treatment has been used to induce S422 phosphorylation in Jurkat cells, as demonstrated in Western blot validation studies .

• While not specific to S422, inhibitors of the PI-3 kinase pathway (such as LY249002) have been shown to affect phosphorylation of CTBP1 at other sites , suggesting that signaling pathway manipulation could affect S422 phosphorylation indirectly.

• Since HIPK2 phosphorylates CTBP1 at S422, activators or inhibitors of HIPK2 would likely modulate S422 phosphorylation.

• The role of CTBP1 in metabolic syndrome suggests that metabolic stressors might influence its phosphorylation status .

Researchers should design time-course experiments to determine optimal treatment durations for studying S422 phosphorylation dynamics.

How does CTBP1 S422 phosphorylation contribute to metabolic syndrome and polycystic ovary syndrome (PCOS)?

CTBP1 expression is significantly elevated in primary granulosa cells (pGCs) derived from PCOS patients with metabolic syndrome and positively correlates with serum triglyceride levels while negatively correlating with serum estradiol (E2) and high-density lipoprotein levels .

Although the specific role of S422 phosphorylation in this context isn't directly addressed in the search results, the mechanistic study revealed that CTBP1 physically binds to the promoter II of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) to inhibit aromatase gene transcription and expression, resulting in reduced E2 synthesis . Additionally, CTBP1 interacts with phosphorylated SREBP1a at S396 in nuclei, leading to FBXW7-dependent protein degradation and reduced lipid droplet formation in pGCs .

Given that S422 phosphorylation regulates CTBP1 stability through proteasomal degradation, it likely represents a critical regulatory point that influences these CTBP1-mediated effects on hormone synthesis and lipid metabolism. Researchers studying metabolic disorders should consider investigating S422 phosphorylation status in relevant cell types.

What is the relationship between CTBP1 S422 phosphorylation and cancer development?

CTBP1 has been identified as an emerging oncogene and potential drug target . While the search results don't explicitly connect S422 phosphorylation to cancer development, several lines of evidence suggest its importance:

• CTBP proteins function as transcriptional co-repressors that can be targeted through therapeutic peptides capable of disrupting CTBP-mediated transcriptional repression in cancer models .

• Disruption of CTBP1-mediated transcriptional repression can reverse CTBP1-mediated oncogenic phenotypes in melanoma models both in cell culture and in mice .

• Since S422 phosphorylation regulates CTBP1 degradation, alterations in this phosphorylation could potentially affect CTBP1 protein levels and its oncogenic functions.

Researchers could investigate whether cancer cells show altered patterns of S422 phosphorylation compared to normal cells, and whether manipulation of this phosphorylation impacts cancer cell phenotypes.

How does S422 phosphorylation interplay with other post-translational modifications of CTBP1?

CTBP1 undergoes multiple post-translational modifications including:

• Phosphorylation at various sites (S158, T176, S300, S422)

• ADP-ribosylation when cells are exposed to brefeldin A

• Sumoylation on Lys-428, promoted by the E3 SUMO-protein ligase CBX4

The potential interplay between these modifications presents a complex regulatory network. For example, phosphorylation at S158 by Pak1 occurs preferentially when CTBP1 is bound to NADH and blocks its dehydrogenase activity . Researchers should consider using multiple modification-specific antibodies in parallel experiments to determine whether these modifications occur sequentially, simultaneously, or mutually exclusively. Mass spectrometry approaches could also help identify patterns of multiple modifications on the same CTBP1 molecule.

What is the temporal dynamics of CTBP1 S422 phosphorylation during cell cycle progression?

The search results indicate that "the level of phosphorylation appears to be regulated during the cell cycle" , although specific details about S422 phosphorylation dynamics are not provided. To characterize these dynamics, researchers could:

• Synchronize cells at different cell cycle stages (G1, S, G2/M) using established methods such as double thymidine block or nocodazole treatment

• Analyze S422 phosphorylation levels at these different stages using the phospho-specific antibody

• Correlate phosphorylation patterns with cell cycle markers

• Investigate the consequences of disrupting this phosphorylation on cell cycle progression

Such studies would elucidate whether S422 phosphorylation serves as a cell cycle checkpoint mechanism or influences CTBP1 functions in a cell cycle-dependent manner.

How can researchers distinguish between CTBP1 and CTBP2 phosphorylation in experimental systems?

CTBP1 and CTBP2 are closely related proteins with some overlapping functions and similar phosphorylation sites. The search results indicate that some antibodies detect phosphorylation at analogous sites in both proteins (e.g., Phospho-CTBP1/CTBP2 (Ser158, Ser164) antibodies) . To distinguish between them:

• Use antibodies specifically developed against unique peptide sequences surrounding the phosphorylation sites in each protein

• Perform immunoprecipitation with isoform-specific antibodies followed by phospho-specific detection

• Use genetic approaches such as selective knockout or knockdown of either CTBP1 or CTBP2

• Consider the differential tissue distribution of CTBP1 versus CTBP2

• Use mass spectrometry to identify isoform-specific peptides containing the phosphorylated residue

Researchers should be particularly careful when interpreting results from antibodies that recognize both proteins and should include appropriate controls.

What are the most common technical challenges when using Phospho-CTBP1 (S422) antibodies and how can they be addressed?

Based on the antibody information provided, researchers might encounter several challenges:

Additionally, researchers should confirm antibody lot consistency by requesting validation data from suppliers when purchasing new lots.

How does sample preparation affect the detection of S422 phosphorylation?

Proper sample preparation is critical for accurate detection of phosphorylated proteins:

• Phosphatase inhibitors: Always include a comprehensive phosphatase inhibitor cocktail in lysis buffers to prevent dephosphorylation during sample preparation.

• Lysis conditions: Use lysis buffers compatible with phosphoprotein preservation (e.g., those containing sodium fluoride, sodium orthovanadate, β-glycerophosphate).

• Sample handling: Process samples quickly and keep them cold to minimize dephosphorylation.

• Protein denaturation: For Western blotting, complete denaturation is critical; the search results mention using 6M guanidine HCl for purification of histidine-tagged CTBP1 when studying phosphorylation .

• Fixation for IHC/IF: Phospho-epitopes can be sensitive to fixation conditions; validate optimal fixation protocols (paraformaldehyde concentration and duration) for your specific samples.

Additionally, researchers should consider fractionation approaches (nuclear vs. cytoplasmic) as CTBP1 localization can change with its phosphorylation status, potentially affecting detection sensitivity in different cellular compartments.

What high-throughput methods can be used to study CTBP1 S422 phosphorylation dynamics in complex biological systems?

For comprehensive analysis of S422 phosphorylation across biological systems, researchers could consider:

• Phosphoproteomics using mass spectrometry: This approach can identify and quantify S422 phosphorylation across multiple samples without antibody limitations.

• Reverse Phase Protein Arrays (RPPA): When validated with phospho-specific antibodies, this method allows high-throughput analysis of phosphorylation across many samples.

• Single-cell phospho-flow cytometry: Enables analysis of phosphorylation heterogeneity across cell populations.

• Biosensor development: Creation of FRET-based biosensors specifically designed to monitor S422 phosphorylation dynamics in live cells.

• Spatial proteomics techniques: Combining phospho-specific antibodies with multiplexed imaging methods like CODEX or CyCIF to study phosphorylation patterns in tissue contexts.

These approaches would enable researchers to understand S422 phosphorylation beyond traditional techniques like Western blotting and immunohistochemistry, potentially revealing new biological insights.

How can researchers utilize CTBP1 S422 phosphorylation status as a biomarker for disease states?

The connection between CTBP1 and conditions like PCOS with metabolic syndrome or cancer suggests potential for S422 phosphorylation as a biomarker:

• Development of tissue microarray analysis using phospho-S422 antibodies to screen patient samples across different disease states.

• Correlation studies between S422 phosphorylation levels and clinical outcomes in diseases where CTBP1 plays a role.

• Liquid biopsy approaches to detect phosphorylated CTBP1 in circulating extracellular vesicles as potential non-invasive biomarkers.

• Investigation of S422 phosphorylation in response to treatments, potentially serving as a pharmacodynamic biomarker.

• Combination with other CTBP1 modifications to develop a "CTBP1 modification signature" with improved biomarker specificity.

Such applications would require extensive validation of antibody specificity and correlation with established disease markers before clinical implementation.

What are the potential off-target effects of CTBP inhibitors and how can they be assessed using phospho-specific antibodies?

Recent research into CTBP inhibitors like MTOB and 4-Cl-HIPP has raised questions about their specificity . Phospho-specific antibodies can help address these concerns:

• Researchers can use phospho-S422 and other site-specific antibodies to determine whether these inhibitors affect CTBP1 phosphorylation status, potentially indicating pathway-specific effects.

• Comparing phosphorylation patterns between wildtype and CTBP1/2 double knockout cell lines treated with inhibitors can help distinguish on-target from off-target effects.

• Time-course and dose-response studies with phospho-specific detection can reveal the kinetics of inhibitor effects on CTBP1 regulation.

• Correlation between changes in phosphorylation status and phenotypic outcomes can help determine which effects are mediated through CTBP1 regulation versus other mechanisms.