SIAH2 Antibody, HRP conjugated

What is SIAH2 and what cellular processes does it regulate?

SIAH2 (Seven In Absentia Homolog 2) is an E3 ligase that catalyzes ubiquitination and proteasome-mediated degradation of protein substrates. It encodes a 324 amino acid protein that shares 77% identity with human SIAH1 and 68% identity with the Drosophila sina (seven in absentia) gene, which is critical for the development of Drosophila R7 photoreceptor . SIAH2 regulates multiple cellular processes through targeted degradation of specific proteins. Under stress conditions such as hypoxia, SIAH2 targets TRAF2 for degradation, which influences cell responses to stress and cytokines through regulation of key stress-signaling cascades . It also targets HIF-1α prolyl hydroxylase 3 (PHD3) for degradation upon exposure to hypoxic conditions, coinciding with increased SIAH2 transcription . Additionally, SIAH2 can decrease TNF-α-dependent induction of JNK activity and transcriptional activation of NFκB, influencing inflammatory responses . Studies with SIAH2 null mice subjected to hypoxia have demonstrated an impaired respiratory response and reduced hemoglobin levels, indicating its physiological importance .

What applications is SIAH2 Antibody, HRP conjugated suitable for?

SIAH2 Antibody, HRP conjugated is a versatile research tool suitable for multiple applications in molecular and cellular biology. It can be effectively used for Western blotting (WB), enabling direct detection of SIAH2 protein in cell or tissue lysates without requiring a secondary antibody . The antibody is also validated for immunohistochemistry on paraffin-embedded sections (IHC-P), allowing visualization of SIAH2 distribution in tissue samples . Additionally, it works effectively in immunofluorescence (IF) applications for studying subcellular localization of SIAH2 . The HRP conjugation eliminates the need for secondary antibody incubation, reducing background signal and cross-reactivity issues while streamlining experimental protocols. This makes it particularly valuable for detecting SIAH2 in complex experimental systems where minimizing procedural variables is important for consistent results.

What species reactivity does SIAH2 Antibody, HRP conjugated demonstrate?

SIAH2 Antibody (24E6H3) HRP demonstrates reactivity against SIAH2 from multiple species, making it a versatile tool for comparative studies across different experimental models. It efficiently detects SIAH2 of human, mouse, and rat origin . This cross-species reactivity is particularly valuable for researchers working with various model systems, as it enables direct comparison of SIAH2 expression and function across species without changing detection reagents. The ability to maintain consistent reagent usage across different model organisms enhances experimental reproducibility and facilitates translational research between animal models and human studies. This multi-species reactivity has been validated through Western blot, immunofluorescence, and immunohistochemistry applications, confirming its utility across various experimental platforms.

How does SIAH2 regulate the ubiquitin-proteasome pathway in hypoxic conditions?

SIAH2 plays a critical role in hypoxia response through strategic regulation of the ubiquitin-proteasome pathway. Under hypoxic conditions, SIAH2 transcription increases, leading to enhanced targeting of specific proteins for ubiquitination and subsequent degradation . One key mechanism involves SIAH2-mediated degradation of HIF-1α prolyl hydroxylase 3 (PHD3), which prevents hydroxylation of HIF-1α and thereby stabilizes it, enabling activation of hypoxia-responsive genes . Additionally, SIAH2 directly ubiquitinates DBC1 at Lysine 287 (K287) under hypoxic stress, leading to its proteasomal degradation . This ubiquitination occurs through K48-linked polyubiquitin chains, the canonical signal for proteasomal targeting . Experiments using proteasome inhibitor MG132 demonstrated complete reversal of DBC1 destabilization, while lysosomal inhibitor bafilomycin A1 had no effect, confirming that SIAH2-mediated DBC1 degradation occurs specifically through the proteasome pathway rather than lysosomes . Cell-based studies have shown that deletion of SIAH2 attenuates the reduction of p53 pathway activity under hypoxia and inhibits cell growth and migration, highlighting the functional significance of this pathway in cellular adaptation to low oxygen conditions .

What is the structural basis for SIAH2 interaction with DBC1?

The interaction between SIAH2 and DBC1 involves specific structural domains that facilitate targeted ubiquitination. Co-immunoprecipitation (Co-IP) assays have demonstrated that exogenously expressed Flag-SIAH2RM (an SIAH2 enzyme-inactive mutant) and Myc-DBC1 reciprocally co-immunoprecipitate, confirming their physical interaction . Importantly, endogenous SIAH2 and DBC1 also interact in cells, and purified glutathione S-transferase (GST) SIAH2 directly pulls down His-DBC1 in vitro, indicating a direct protein-protein interaction rather than one mediated by additional factors . Domain mapping studies using truncated constructs revealed that amino acids 1-230 at the N-terminus of DBC1 are necessary for binding to SIAH2 . Conversely, the full-length SIAH2 protein is required for this interaction, as truncated versions fail to efficiently bind DBC1 . Pull-down experiments with purified proteins confirmed that the N-terminus (1-461) of DBC1 interacts with SIAH2, while the C-terminus (462-923) does not . This structural interaction provides the molecular basis for SIAH2's ability to specifically ubiquitinate DBC1 at Lysine 287, as identified through mass spectrometry analysis . Mutation of this lysine residue (K287R) prevents SIAH2-mediated ubiquitination and subsequent degradation of DBC1, confirming the specificity of this interaction .

How can I optimize SIAH2 Antibody, HRP conjugated for detection in challenging tissue samples?

Optimizing SIAH2 Antibody, HRP conjugated for difficult tissue samples requires systematic modification of several protocol parameters. For effective antigen retrieval, compare different buffer systems (citrate pH 6.0 versus EDTA pH 8.0 versus Tris-EDTA pH 9.0) and methods (pressure cooker, microwave, or water bath) . The optimal conditions will vary depending on fixation, tissue type, and processing methods. For samples with high background or weak signal, implementing signal amplification using tyramide signal amplification (TSA) systems can significantly enhance detection sensitivity while maintaining specificity . Background reduction is crucial and should include thorough blocking of endogenous peroxidase activity using 3% H₂O₂ for 10-30 minutes followed by protein blocking with 5% normal serum or 1% BSA for 30-60 minutes . For tissues with high biotin content, include avidin/biotin blocking steps. Tissue-specific modifications may be necessary - for example, hypoxic tumor regions might require comparison with HIF-1α staining as a reference for expected SIAH2 localization patterns . Always include appropriate controls: SIAH2 knockout or siRNA-treated samples can serve as negative controls, while tissues known to express high SIAH2 levels (such as hypoxic tumor regions) provide positive controls . Document optimization steps systematically to establish reproducible staining protocols for your specific applications.

What methods can be used to quantify SIAH2-dependent ubiquitination activity?

Several complementary approaches can be employed to quantify SIAH2-dependent ubiquitination activity with high specificity. In vitro ubiquitination assays provide a defined system for direct measurement using purified components including recombinant SIAH2, E1/E2 enzymes, ubiquitin, ATP, and purified substrate proteins like DBC1 . The ubiquitinated products can be detected via Western blot and quantified through densitometry. For cell-based systems, co-transfection of SIAH2 and substrate proteins (e.g., DBC1) followed by treatment with proteasome inhibitors (MG132, 10μM, 4-6 hours) prevents degradation of ubiquitinated products, allowing their accumulation and detection . Immunoprecipitation of substrate proteins followed by Western blotting with anti-ubiquitin antibodies reveals the extent of ubiquitination. For studying specific ubiquitin chain types, K48-specific ubiquitin antibodies can be used to quantify proteasome-targeting chains, as demonstrated in studies showing SIAH2 induces K48-linked polyubiquitination of DBC1 . The cycloheximide chase assay provides a functional readout of SIAH2 activity by measuring substrate protein half-life in the presence or absence of active SIAH2 . For all methods, comparison between wildtype SIAH2 and the enzyme-inactive mutant (SIAH2RM) serves as an essential control to confirm specificity . These approaches collectively enable comprehensive assessment of SIAH2 E3 ligase activity in various experimental contexts.

What is the clinical significance of SIAH2 expression in cancer progression?

SIAH2 expression has significant clinical relevance in cancer progression, particularly in breast cancer. Analysis of The Cancer Genome Atlas (TCGA) data reveals that SIAH2 expression positively correlates with tumor stage and number of lymph nodes affected, both key indicators of tumor malignancy and progression . Comparative studies between cancer tissues and normal samples have demonstrated that SIAH2 is significantly upregulated in breast cancer tissues . Importantly, tissue microarray analysis of breast cancer patient samples shows a negative correlation between SIAH2 and DBC1 expression, consistent with the molecular mechanism whereby SIAH2 targets DBC1 for degradation . Functionally, deletion of SIAH2 in breast cancer cell lines attenuates the reduction of p53 pathway activity under hypoxia and promotes etoposide-triggered cell apoptosis, effects that can be rescued by simultaneous knockout of CCAR2 (DBC1) . This indicates that the SIAH2-DBC1 axis directly influences cancer cell survival pathways. In vivo studies with animal models have further confirmed that SIAH2 contributes to tumorigenesis and tumor progression through these mechanisms . These findings collectively define a key role for the hypoxia-mediated SIAH2-DBC1 pathway in breast cancer progression and suggest potential therapeutic approaches targeting this pathway .

What are optimal protocols for using SIAH2 Antibody, HRP conjugated in multiplex immunohistochemistry?

Implementing SIAH2 Antibody, HRP conjugated in multiplex immunohistochemistry requires careful protocol design to maintain specificity while enabling detection of multiple targets. For sequential tyramide-based multiplex approaches, apply SIAH2 Antibody, HRP conjugated (typically at 1:100-1:200 dilution) followed by development with tyramide-conjugated fluorophores (e.g., Tyramide-Cy3) for 10 minutes . After signal development, perform microwave treatment (10 minutes) to denature bound antibodies while preserving the covalently-linked tyramide signal. Block with 5% normal serum (30 minutes) before applying the next primary antibody and its respective detection system . For chromogenic multiplexing, use SIAH2 Antibody, HRP with one chromogen (e.g., DAB, brown), perform antibody stripping (glycine-SDS pH 2.0, 10 minutes), block with avidin/biotin if using biotin-based detection, then apply the next antibody with an alternative chromogen (e.g., AP-Red) . When designing multiplex panels, include markers relevant to SIAH2 function such as HIF-1α, DBC1, and p53 to provide mechanistic context . To prevent cross-reactivity, employ thorough blocking between steps (10% normal serum from subsequent antibody species) and complete HRP inactivation (3% H₂O₂, 15 minutes) . For automated platforms, program appropriate antigen retrieval for all targets (typically HIER pH 9.0) and incorporate extended washing steps between antibodies (3x5 minutes TBST) . Validate specificity using single-stained controls alongside multiplex samples to confirm signal specificity.

What strategies can minimize non-specific binding when using SIAH2 Antibody, HRP conjugated?

Minimizing non-specific binding when using SIAH2 Antibody, HRP conjugated requires implementation of multiple optimization strategies. Extended blocking protocols with 5% BSA or 5-10% normal serum (1-2 hours at room temperature) can significantly reduce non-specific interactions . For challenging samples, commercial protein-free blocking solutions may provide superior results. Adding 0.1-0.3% Triton X-100 to blocking buffer improves penetration and reduces hydrophobic interactions. Buffer optimization is equally important - include 0.05-0.1% Tween-20 in all wash and antibody diluent buffers and consider adding 0.1-0.5M NaCl to reduce ionic interactions . For tissues with high endogenous peroxidase activity, extend H₂O₂ treatment (0.3-3%, 20-30 minutes) to ensure complete quenching before antibody application . Antibody dilution optimization is critical - test multiple dilutions to identify optimal concentration (typically 1:100-1:1000) that provides specific signal with minimal background. Consider extending primary antibody incubation time at 4°C (overnight) with more dilute antibody solutions . Always prepare fresh dilutions of HRP-conjugated antibody for each experiment and implement thorough detergent wash steps (3-5 washes, 5 minutes each) between incubations. For Western blotting applications specifically, include 1-5% non-fat dry milk in blocking solutions to reduce membrane-associated background . Document background levels systematically under different conditions to identify optimal protocol parameters for your specific experimental system.

How can I use SIAH2 Antibody, HRP conjugated to study hypoxia-induced protein degradation?

SIAH2 Antibody, HRP conjugated provides a valuable tool for studying hypoxia-induced protein degradation pathways. To establish experimental systems, culture cells under controlled hypoxic conditions (1-2% O₂) using specialized hypoxia chambers or chemical hypoxia mimetics such as cobalt chloride (CoCl₂) or dimethyloxalylglycine (DMOG) . Monitor SIAH2 protein levels via Western blotting with the HRP-conjugated antibody, which typically shows increased expression under hypoxic conditions coinciding with its role in hypoxic adaptation . To study SIAH2-substrate interactions during hypoxia, perform co-immunoprecipitation experiments using SIAH2 Antibody to pull down protein complexes followed by Western blot analysis of known or suspected substrates such as DBC1 or PHD3 . For studying substrate degradation kinetics, conduct cycloheximide chase experiments under normoxic versus hypoxic conditions while tracking substrate protein levels over time (typically 0-8 hours) . To directly assess ubiquitination, perform ubiquitination assays by immunoprecipitating substrate proteins (e.g., DBC1) followed by Western blotting with ubiquitin antibodies, comparing normoxic versus hypoxic samples . Include key controls such as proteasome inhibitors (MG132, 10μM) to block degradation and allow accumulation of ubiquitinated intermediates . For spatial analysis, perform immunohistochemistry on serial sections of hypoxic tissues (such as tumor samples) to correlate SIAH2 expression with hypoxia markers (e.g., HIF-1α) and substrate proteins . These approaches collectively enable comprehensive investigation of SIAH2-mediated protein degradation under hypoxic stress conditions.

How do I interpret conflicting results between SIAH2 protein levels and ubiquitination activity?

Interpreting discrepancies between SIAH2 protein levels and ubiquitination activity requires consideration of multiple regulatory mechanisms. First, post-translational modifications of SIAH2 itself, including phosphorylation, can alter its E3 ligase activity without changing protein levels . Second, hypoxic conditions increase SIAH2 transcription while simultaneously enhancing its activity, potentially creating temporal differences between protein expression and functional outcomes . Third, the presence of competing substrate proteins can influence observed ubiquitination patterns for specific targets like DBC1 . Fourth, contextual factors such as cell type, tissue microenvironment, and experimental conditions (acute versus chronic hypoxia) can significantly impact SIAH2 function . To resolve these discrepancies, implement multiple complementary assays: (1) measure both total SIAH2 protein and phosphorylated SIAH2 to assess activation state; (2) directly quantify E3 ligase activity using in vitro ubiquitination assays with purified components; (3) compare wildtype SIAH2 with the enzyme-inactive mutant (SIAH2RM) to distinguish between expression and activity effects; (4) assess SIAH2 subcellular localization, which may influence access to specific substrates . Additionally, conduct time-course experiments to determine whether discrepancies reflect temporal differences in protein expression versus activation. Always collect both functional (ubiquitination, substrate degradation) and expression (protein levels) data within the same experimental system to enable direct comparisons and facilitate accurate interpretation of complex regulatory relationships.

What troubleshooting approaches can resolve weak or non-specific signals with SIAH2 Antibody, HRP conjugated?

When encountering weak or non-specific signals with SIAH2 Antibody, HRP conjugated, systematic troubleshooting approaches can identify and resolve the underlying issues. For weak signals, first verify protein expression - SIAH2 levels may be naturally low in certain cell types or conditions, requiring loading more total protein (50-100 μg) for Western blots . Enhance signal detection by implementing more sensitive substrates such as enhanced chemiluminescence (ECL) reagents or increasing exposure time . For immunohistochemistry applications, optimize antigen retrieval conditions by testing different buffer systems and methods as detailed in question 2.3 . Consider signal amplification approaches such as tyramide signal amplification (TSA) systems, which can significantly boost detection sensitivity . For non-specific signals in Western blotting, first verify sample quality by assessing protein degradation with Ponceau staining. Optimize blocking conditions by testing different blockers (BSA, non-fat milk, commercial blockers) and increase blocking time to 1-2 hours . Include 0.1-0.5% Tween-20 in wash buffers and antibody diluents to reduce hydrophobic interactions. For multiple bands, assess whether they represent different isoforms, post-translationally modified forms, or degradation products by comparing with positive and negative control samples . If background persists in immunohistochemistry applications, implement stringent washing steps (3-5 washes, 5 minutes each) and consider more dilute antibody preparations with longer incubation times (overnight at 4°C) . For all applications, validate antibody specificity using the approaches outlined in question 3.2 to confirm that observed signals represent genuine SIAH2 detection.

How do research findings on SIAH2-DBC1 interaction compare across different experimental models?

Research findings on the SIAH2-DBC1 interaction show remarkable consistency across different experimental models while revealing context-specific nuances. In cell-based systems, exogenously expressed Flag-SIAH2RM and Myc-DBC1 reciprocally co-immunoprecipitate, and this interaction is confirmed between endogenous proteins in multiple cell lines . Biochemical studies with purified proteins demonstrate direct interaction between GST-SIAH2 and His-DBC1 in vitro, confirming that their association does not require additional cellular factors . Structurally, the interaction involves the N-terminus (amino acids 1-230) of DBC1 binding to full-length SIAH2 across different experimental systems . Functionally, SIAH2 consistently ubiquitinates DBC1 at Lysine 287 in various cell types, leading to proteasome-dependent degradation that is blocked by MG132 but not by lysosomal inhibition with bafilomycin A1 . The hypoxia-dependent nature of this interaction is conserved across different cell lines, with hypoxic conditions enhancing SIAH2-mediated DBC1 degradation . In animal models, SIAH2 knockout demonstrates impaired hypoxic response, consistent with its role in cellular adaptation . Moving to human clinical samples, tissue microarray analysis of breast cancer patients shows a negative correlation between SIAH2 and DBC1 expression, matching the molecular mechanism established in cellular and biochemical studies . These findings collectively demonstrate a robust and conserved SIAH2-DBC1 regulatory pathway that functions across experimental models from purified proteins to human clinical samples, validating its biological significance in hypoxic response and cancer progression.

What are the emerging research directions for SIAH2 Antibody applications?

Emerging research directions for SIAH2 Antibody applications span multiple frontiers in cancer biology, cellular stress responses, and therapeutic development. Single-cell analysis represents a promising avenue, with SIAH2 antibodies being adapted for mass cytometry (CyTOF) and imaging mass cytometry to map SIAH2 expression patterns at single-cell resolution within heterogeneous tissues, particularly tumors with varying hypoxic regions . Spatial transcriptomics combined with SIAH2 protein detection offers opportunities to correlate protein expression with transcriptional programs across tissue microenvironments . The development of structure-based Siah inhibitors represents another frontier, with SIAH2 antibodies serving as critical tools for validating target engagement and efficacy in both in vitro and in vivo models . The SIAH2-DBC1 axis in breast cancer progression offers opportunities for biomarker development, potentially using SIAH2/DBC1 expression ratios as prognostic indicators . Beyond cancer, SIAH2's role in hypoxic adaptation suggests applications in cardiovascular disease, stroke, and high-altitude physiology research . Methodologically, adapting SIAH2 antibodies for live-cell imaging through development of cell-permeable antibody fragments could enable real-time monitoring of SIAH2 dynamics during hypoxic response . Additionally, combining SIAH2 detection with other ubiquitin pathway components through multiplex approaches will provide more comprehensive understanding of how E3 ligase networks coordinate responses to cellular stress . These diverse research directions highlight the continuing importance of SIAH2 antibodies as versatile tools for understanding fundamental biological processes and developing novel therapeutic approaches.

How can SIAH2 Antibody, HRP conjugated contribute to therapeutic development?

SIAH2 Antibody, HRP conjugated provides essential capabilities for therapeutic development targeting the SIAH2 pathway. For target validation, the antibody enables confirmation of SIAH2 expression in patient-derived samples, helping identify cancer types most likely to respond to SIAH2-targeting therapies . In drug discovery, it facilitates high-throughput screening assays to identify compounds that modulate SIAH2 activity or expression, with structure-based design of covalent Siah inhibitors representing a promising approach . For monitoring drug efficacy, the antibody allows assessment of SIAH2 inhibition in cellular and animal models through Western blotting and immunohistochemistry of treated samples . In biomarker development, SIAH2 detection combined with analysis of its substrates like DBC1 could define signature patterns predictive of treatment response, particularly in hypoxic tumors that are often therapy-resistant . The relationship between SIAH2 and breast cancer progression suggests potential applications in companion diagnostics, where SIAH2/DBC1 expression patterns might predict response to targeted therapies . For pharmacodynamic studies, the antibody enables monitoring of downstream pathway effects following drug treatment, such as changes in substrate protein levels or activation of p53 pathways . The ability to detect SIAH2 across multiple species (human, mouse, rat) facilitates translation between preclinical models and human studies . Additionally, multiplex approaches combining SIAH2 detection with other cancer biomarkers can provide comprehensive characterization of tumor biology to guide combination therapy strategies . These diverse applications position SIAH2 Antibody, HRP conjugated as a valuable tool along multiple stages of the therapeutic development pipeline, from target discovery through clinical application.