SERTAD1 Antibody

What is SERTAD1 and what are its key characteristics?

SERTAD1 (SERTA domain containing 1, also known as SEI1 or TRIP-Br1) is a 25 kDa protein that functions as a cyclin-dependent kinase 4 (cdk4)-binding protein. It acts as a growth factor sensor and facilitates the formation and activation of cyclin D-CDK complexes when faced with inhibitory levels of INK4 proteins. SERTAD1 antagonizes the activity of the p16(INK4a) tumor suppressor and suppresses CREB-mediated transcription. Additionally, it regulates cell cycle progression through sequential effects on the transcriptional activity of E2F-responsive promoters during G1 and S phases .

SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. It does not contain any known DNA binding motifs and has not been reported to interact with DNA directly. Instead, it forms complexes with transcription factors like E2F1 to co-activate the transcription of target genes .

What are the key specifications of commercially available SERTAD1 antibodies?

Commercial SERTAD1 antibodies (such as 10167-1-AP) are typically available with the following specifications:

These antibodies are primarily designed for immunohistochemistry (IHC) and ELISA applications .

What is the recommended protocol for using SERTAD1 antibody in immunohistochemistry?

For immunohistochemistry applications, SERTAD1 antibody should be used at a dilution range of 1:50-1:500, with specific dilution optimized based on sample type and experimental conditions. Antigen retrieval is recommended with TE buffer pH 9.0, though citrate buffer pH 6.0 can serve as an alternative. Positive IHC signals have been detected in mouse heart tissue and mouse embryo tissue .

It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as the ideal dilution may be sample-dependent. For cellular localization studies, research has shown that SERTAD1 is detectable in both nuclear and cytoplasmic fractions, with localization not significantly altered by BMP stimulation .

How should SERTAD1-SMAD1 interactions be detected in experimental settings?

Multiple complementary approaches can be used to detect SERTAD1-SMAD1 interactions:

• Yeast two-hybrid screening: Using SMAD1 as bait, researchers have successfully identified SERTAD1 as an interaction partner. Positive interaction is indicated by yeast cells harboring both SERTAD1 prey construct and SMAD1 bait construct growing on selective medium .

• In vitro GST pull-down analysis: This technique confirms direct protein-protein interactions without mediation by other cellular factors. 35S-labeled SERTAD1 can be pulled down with GST-SMAD1 but not with GST alone, demonstrating direct interaction between the two proteins .

• Mammalian cell GST pull-down analysis: Co-express GST-SERTAD1 (or GST alone as negative control) and HA-SMAD1 in HEK293 cells, potentially with a constitutively active form of ALK6 (Q203D) to activate the BMP intracellular signaling cascade. HA-SMAD1 will co-purify with GST-SERTAD1 but not with GST from cell lysates, confirming interaction in a mammalian cellular context .

• Phosphorylation-specific detection: Active (phosphorylated) SMAD1 can be co-purified with SERTAD1 and detected through western analysis using a p-SMAD1 specific antibody, indicating functional significance of the interaction .

What methods are available for analyzing SERTAD1 expression in tissue samples?

Several complementary methods can be employed for comprehensive SERTAD1 expression analysis:

• RT-PCR analysis: Total RNA should be isolated from target tissues (e.g., embryonic hearts) using an RNA isolation kit (such as RNeasy mini kit). OneStep RT-PCR can then be performed to detect SERTAD1 mRNA expression .

• Western blot analysis: This approach detects SERTAD1 protein expression levels, with the expected molecular weight of 25 kDa. For subcellular localization studies, nuclear/cytoplasmic fractionation can be performed prior to western analysis using markers such as MEK1/2 (cytoplasmic marker) and LSD1 (nuclear marker) to validate fractionation quality .

• In situ hybridization: Non-isotope section in situ analysis can be performed using a probe derived from the 3' untranslated region of SERTAD1 (~300bp-cDNA fragment). This allows visualization of spatial SERTAD1 expression patterns in tissues .

• Immunostaining: For cellular localization, culture cells on glass coverslips and incubate with SERTAD1 primary antibody at 4°C overnight, followed by incubation with fluorescent-conjugated secondary antibody. Counterstain with DAPI to visualize nuclei and examine using confocal microscopy .

How should functional analysis of SERTAD1 transcriptional co-activation be performed?

To assess SERTAD1's role as a transcriptional co-activator, researchers can implement these approaches:

• Reporter assays: Co-transfect cells (such as NkL-TAg cardiomyocytes) with a BMP-responsive reporter construct and different doses of a construct expressing SERTAD1. Luciferase activity can be measured to determine SERTAD1's effect on transcriptional activity. This approach has demonstrated that SERTAD1 enhances the activity of BMP reporters in a dose-dependent manner .

• Gene expression analysis: After SERTAD1 overexpression in cardiomyocytes, analyze the expression of known BMP/SMAD regulatory targets (such as NKX2.5, ID2, and TBX20) using RT-PCR or qPCR to determine if SERTAD1 upregulates their expression upon BMP stimulation .

• BMP stimulation experiments: Compare baseline expression versus BMP-stimulated expression in the presence or absence of SERTAD1 overexpression. This can help elucidate SERTAD1's role in the BMP signaling pathway .

What is the role of SERTAD1 in cardiogenesis and heart development?

SERTAD1 plays a significant role in heart development through its function as a SMAD1 transcriptional co-activator in the BMP signaling pathway. Research has shown that SERTAD1 is expressed in developing hearts, and its interaction with SMAD1 promotes the expression of BMP target genes during mouse cardiogenesis .

The subcellular localization analysis has demonstrated that SERTAD1 is present in both the cytoplasm and nucleus of cardiomyocytes. The nuclear localization of SERTAD1 and its interaction with phosphorylated SMAD1 supports its role as a transcriptional co-activator. Functional analysis has shown that overexpression of SERTAD1 in immortalized cardiomyocytes enhances the activities of BMP-responsive reporters and upregulates the expression of endogenous cardiac development genes including NKX2.5, ID2, and TBX20 upon BMP stimulation .

These findings establish SERTAD1 as an important factor in the molecular pathways governing heart development, specifically through the modulation of BMP signaling, which is critical for proper cardiac morphogenesis.

What is the prognostic significance of SERTAD1 in cancer research?

Specifically, heterogeneity analysis in fixed effect models has shown significant correlations between SERTAD1 expression and various survival metrics:

These statistical analyses provide robust evidence of an association between SERTAD1 expression levels and cancer outcomes, suggesting its potential utility as a biomarker for cancer prognosis and potential therapeutic target.

How does SERTAD1 interact with cell cycle regulation pathways?

SERTAD1 plays critical roles in cell cycle regulation through several mechanisms:

• CDK4 interaction: SERTAD1 directly interacts with cyclin-dependent kinase 4 (CDK4) to antagonize the activity of the cyclin-dependent kinase inhibitor p16INK4a, thereby promoting cell proliferation .

• E2F transcription factor regulation: SERTAD1 forms complexes with E2F transcription factors to co-activate the transcription of target genes involved in cell cycle progression. It also negatively regulates E2F transcription factor by modulating p16INK4a during the G1/S phase of the cell cycle .

• Retinoblastoma pathway involvement: SERTAD1 interacts with the retinoblastoma protein pathway (pRb1-cyclinD1-cdk4/6-p16INK4A), which is crucial for cell cycle control. Additionally, the retinoblastoma gene (RB) and adenovirus E1A oncogene modulate the action of E2F-1/DP-1 and SERTAD1/KRIP-1 .

• Protein phosphatase interaction: The B-alpha isoform of PP2A (serine/threonine protein phosphatase 2A) controls cell proliferation through an association between PP2A-AB-alphaC holoenzyme and SERTAD1 .

Inhibition of SERTAD1 has been shown to result in cell proliferation arrest in various cancer cell lines, including human nasopharyngeal cancer (CNE2), cervical cancer (CaSki), and melanoma (MeWo) , further supporting its role as a cell cycle modulator.

What are common technical challenges in SERTAD1 immunostaining and how can they be addressed?

When performing immunostaining with SERTAD1 antibody, researchers may encounter several technical challenges:

• Dual localization interpretation: SERTAD1 localizes to both cytoplasm and nucleus, which can complicate interpretation. To address this, always include proper subcellular markers (nuclear and cytoplasmic) as controls. For nuclear/cytoplasmic fractionation, validate with MEK1/2 (cytoplasmic marker) and LSD1 (nuclear marker) .

• Antigen retrieval optimization: SERTAD1 detection is sensitive to antigen retrieval methods. While TE buffer pH 9.0 is recommended, citrate buffer pH 6.0 can be used as an alternative. When signals are weak or absent, systematically test both buffers and vary incubation times to optimize retrieval conditions .

• Signal-to-noise ratio: For clearer distinction between specific staining and background, titrate antibody concentrations carefully (recommended range: 1:50-1:500). Extend blocking steps with appropriate blocking buffers to reduce non-specific binding. Consider signal amplification systems for low-expression samples .

• BMP signaling dependency: When studying SERTAD1-SMAD1 interactions, note that in the absence of active BMP signaling, their association may be marginal. Include positive controls with constitutively active ALK6 to enhance detection of this interaction .

How should researchers approach conflicting SERTAD1 expression data across different experimental models?

When encountering conflicting SERTAD1 expression data across different experimental models:

• Validate antibody specificity: Confirm antibody specificity using positive and negative controls. Consider using siRNA knockdown or CRISPR-Cas9 knockout of SERTAD1 as negative controls to verify antibody specificity in your specific cellular system.

• Consider cell-type specific effects: SERTAD1 expression and function may vary significantly between cell types. For instance, its role in cardiomyocytes related to BMP signaling may differ from its cell cycle regulatory functions in cancer cells . Document the exact cell type and developmental stage used in your experiments.

• Signaling context matters: The SERTAD1-SMAD1 interaction is enhanced through active BMP signaling . Similarly, other SERTAD1 interactions may be context-dependent. Carefully document and control the signaling environment in your experimental system.

• Employ multiple detection methods: When expression data conflicts, use complementary techniques (RT-PCR, western blot, immunostaining, in situ hybridization) to build a more comprehensive picture of SERTAD1 expression patterns .

• Statistical analysis of heterogeneity: When analyzing expression across multiple datasets, employ statistical methods similar to those used in meta-analyses of SERTAD1 in cancer research, which revealed significant heterogeneity (I² values >70%) across different studies and cancer types .

How might SERTAD1 function as a therapeutic target in cancer treatment?

Given SERTAD1's role in cancer progression and its negative correlation with patient survival , several approaches could be explored for targeting SERTAD1 in cancer therapy:

• Direct inhibition strategies: Developing small molecule inhibitors or peptide-based blockers that disrupt SERTAD1's interaction with key partners like CDK4 or SMAD1 could potentially suppress its pro-proliferative effects. Structure-based drug design would require detailed understanding of SERTAD1's functional domains, particularly the PHD-binding domain and C-terminal acidic rich domain that regulate p53-intrinsic transcriptional activities .

• Domain-specific targeting: SERTAD1's functional domains could be selectively targeted. The ablation of specific domains, such as the PHD-binding domain, affects p53-related activities . Therapeutics that mimic this selective inhibition could potentially reduce SERTAD1's oncogenic effects while minimizing off-target impacts.

• Transcriptional regulation: Identifying and modulating transcription factors that control SERTAD1 expression could provide an upstream regulatory approach. Additionally, exploring miRNA regulation of SERTAD1 might reveal opportunities for miRNA-based therapies.

• Pathway intersection targeting: SERTAD1 intersects with multiple cancer-relevant pathways, including the PI3K/Akt/BRCA1-Abraxas pathway involved in Double minute chromosomes (DMs) formation . Combination therapies targeting SERTAD1 along with these intersecting pathways might enhance therapeutic efficacy.

• Biomarker-guided therapy: The strong prognostic significance of SERTAD1 suggests its potential as a biomarker for patient stratification, enabling more personalized treatment approaches for patients with high SERTAD1 expression.

What is the relationship between SERTAD1 and Double Minute Chromosomes (DMs) in cancer genomic instability?

SERTAD1 has been implicated in the formation of Double Minute Chromosomes (DMs), which are associated with chromosomal small fragments and abnormal cellular function in high-grade tumors . The relationship between SERTAD1 and DMs presents several intriguing research questions:

• Mechanistic involvement: SERTAD1 promotes DMs formation through induction of the PI3K/Akt/BRCA1-Abraxas pathway . This suggests a role for SERTAD1 in genomic instability that extends beyond its known transcriptional co-activator functions.

• Met oncogene connection: Research has indicated that SERTAD1 is involved in Met-induced formation of DMs . Further investigation into this relationship could elucidate how growth factor receptor signaling interfaces with genomic instability mechanisms through SERTAD1.

• Correlation with amplified oncogenes: DMs often contain amplified oncogenes critical for cancer progression. Research should explore whether SERTAD1 preferentially influences the amplification of specific oncogenes within DMs structures.

• Prognostic implications: Given that DMs are associated with aggressive cancer phenotypes and poor prognosis, the relationship between SERTAD1 expression, DMs presence, and clinical outcomes merits systematic investigation across cancer types.

• Therapeutic vulnerability: The SERTAD1-DMs relationship may represent a unique therapeutic vulnerability. Disrupting this process could potentially reduce oncogene amplification and genomic instability, thereby limiting cancer progression and therapeutic resistance.

How does the dual cytoplasmic/nuclear localization of SERTAD1 relate to its diverse cellular functions?

The observed dual localization of SERTAD1 in both cytoplasmic and nuclear compartments raises important questions about its functional versatility:

• Compartment-specific interactions: Immunostaining and subcellular fractionation studies have demonstrated SERTAD1's presence in both the cytoplasm and nucleus of cardiomyocytes, with nuclear localization supporting its role as a transcriptional co-activator . Research should identify distinct protein interaction networks in each compartment to understand compartment-specific functions.

• Regulatory mechanisms: The mechanisms controlling SERTAD1's nuclear-cytoplasmic distribution remain poorly understood. Investigation into potential post-translational modifications, binding partners, or signal-dependent shuttling mechanisms would provide insight into this regulatory process. Notably, BMP stimulation does not appear to significantly alter SERTAD1's subcellular distribution , suggesting other regulatory inputs.

• Functional coordination: How SERTAD1's cytoplasmic and nuclear functions are coordinated to regulate cellular processes like cell cycle progression and response to growth factors requires further elucidation. For instance, cytoplasmic SERTAD1 might interact with signaling molecules while nuclear SERTAD1 mediates transcriptional outcomes of the same pathways.

• Therapeutic implications: The dual localization may necessitate compartment-specific targeting strategies for therapeutic interventions. Compounds that selectively disrupt either nuclear or cytoplasmic functions of SERTAD1 might exhibit different effects on cellular phenotypes.

• Cell type specificity: While dual localization has been documented in cardiomyocytes , its localization pattern may vary across cell types and pathological states. Systematic analysis across diverse cellular contexts would help construct a more comprehensive understanding of SERTAD1's biological functions.