SPSB1 Antibody

What is SPSB1 and what are its primary cellular functions?

SPSB1 is a 31 kDa protein (273 amino acids) that functions as a negative regulator of the NF-κB pathway . It belongs to the SPSB family of proteins that contain SOCS box domains, which interact with Cullin-5 (Cul-5) to form E3 ubiquitin ligase complexes. SPSB1 has been shown to suppress NF-κB activation induced by multiple pathways, including those triggered by inflammatory cytokines like IL-1β and TNF-α .

The primary functions of SPSB1 include:

• Suppression of NF-κB-dependent gene expression

• Regulation of inflammatory responses

• Modulation of cellular apoptosis pathways

• Potentiation of c-MET signaling in certain cancer contexts

Methodologically, these functions have been demonstrated through siRNA knockdown experiments, overexpression studies, and immunoprecipitation assays that reveal SPSB1's interaction with key signaling proteins such as p65 .

What experimental applications are suitable for SPSB1 antibodies?

Based on available research data, SPSB1 antibodies can be used in multiple experimental applications:

When designing experiments with SPSB1 antibodies, researchers should consider that optimal dilutions are sample-dependent . The antibody typically recognizes SPSB1 at approximately 31 kDa in western blot applications . For co-immunoprecipitation experiments studying SPSB1 interactions with proteins like p65, it's essential to determine whether stimulation (e.g., with IL-1β) is necessary to enhance the interaction, as studies have shown that pathway activation enhances SPSB1-p65 binding .

How does SPSB1 regulate the NF-κB signaling pathway?

SPSB1 acts as a potent suppressor of NF-κB responses through a mechanism that targets the p65 subunit of NF-κB. Current research indicates that SPSB1 does not affect p65 nuclear translocation or stability, but rather targets its transactivation potential .

The regulatory mechanism involves:

• SPSB1 interaction with p65, which is enhanced after IL-1β treatment

• Requirement of the SOCS-box domain for inhibitory function

• Potential non-degradative ubiquitylation of p65

Studies have shown that SPSB1 depletion results in enhanced NF-κB-dependent transcriptional activity, while its overexpression reverses this effect . Importantly, SPSB1 restricts NF-κB activation induced by both inflammatory cytokines and viral infections.

For researchers studying this pathway, co-immunoprecipitation experiments have confirmed that SPSB1 specifically interacts with p65, and this interaction is significantly enhanced in cells previously treated with IL-1β . The SOCS-box domain of SPSB1, which mediates interaction with Cul-5, is dispensable for binding to p65 but essential for inhibiting p65 transcriptional activity .

How can SPSB1 antibodies be used to study breast cancer recurrence mechanisms?

SPSB1 has been implicated in breast cancer recurrence through its ability to protect cells from apoptosis induced by HER2/neu pathway inhibition or chemotherapy. SPSB1 is spontaneously upregulated during mammary tumor recurrence and is both necessary and sufficient to promote tumor recurrence in genetically engineered mouse models .

Methodological approach for studying SPSB1 in breast cancer recurrence:

• Expression profiling: Use SPSB1 antibodies for immunoblotting to compare SPSB1 expression levels between primary tumors, residual disease, and recurrent tumors.

• Selection mechanism studies: Employ fluorescently labeled SPSB1-expressing cells and control cells to track selective advantage in tumor models before and after therapeutic intervention, as demonstrated in previous research showing SPSB1 cells comprised only 30% of labeled tumor cells in primary tumors but became enriched after HER2/neu downregulation .

• Apoptosis resistance analysis: Combine SPSB1 immunostaining with cleaved caspase-3 detection to evaluate how SPSB1 expression correlates with apoptosis resistance in tumor samples.

• c-MET signaling investigation: Use co-immunoprecipitation with SPSB1 antibodies to study the relationship between SPSB1 and c-MET signaling components, as SPSB1's recurrence-promoting effects have been attributed to its ability to potentiate c-MET signaling .

Previous research has shown that SPSB1-overexpressing tumor cells are selected for following HER2/neu downregulation, and SPSB1 expression is positively correlated with c-MET activity in human breast cancers and with increased risk of relapse in breast cancer patients .

What validation strategies should be employed when using SPSB1 antibodies?

Proper validation of SPSB1 antibodies is crucial for ensuring experimental reliability. Based on research practices, a comprehensive validation approach should include:

• Antibody specificity testing:

  • Western blotting comparing samples with endogenous SPSB1, SPSB1 knockdown, and SPSB1 overexpression

  • Testing reactivity with recombinant SPSB1 protein

  • Comparing multiple antibodies targeting different epitopes of SPSB1

• Knockdown/overexpression controls:

  • Using siRNA or shRNA targeting SPSB1 to confirm antibody specificity

  • Verifying signal reduction in knockdown samples (e.g., using shSPSB1a and shSPSB1b as demonstrated in previous research)

  • Overexpression of tagged SPSB1 constructs to confirm antibody detection

• Cross-reactivity assessment:

  • Testing for potential cross-reactivity with other SPSB family members

  • Evaluating antibody performance across species (human, mouse, rat) as commercial antibodies show reactivity with samples from these species

• Application-specific validation:

  • For immunoprecipitation: Verify enrichment of SPSB1 compared to isotype controls

  • For immunofluorescence: Include subcellular fractionation controls to confirm localization patterns

A systematic validation approach using these methods ensures that experimental observations with SPSB1 antibodies reflect true biological processes rather than technical artifacts.

How can researchers effectively study SPSB1's role in apoptosis resistance mechanisms?

SPSB1 has been shown to protect cells from apoptosis induced by HER2/neu inhibition or chemotherapeutic agents. To effectively study this function, researchers can employ the following methodological approaches:

• Combined knockdown and drug treatment experiments:

  • Use SPSB1 antibodies to confirm knockdown efficiency

  • Treat SPSB1-depleted and control cells with apoptosis-inducing agents

  • Quantify apoptosis through cleaved caspase-3 immunostaining or caspase activity assays

• Clonogenic survival assays:

  • Plate cells with modulated SPSB1 expression levels at low density

  • Expose to apoptosis-inducing treatments

  • Assess colony formation capacity

  • Previous research has demonstrated that SPSB1 cells were far more efficient than control cells at forming colonies in clonogenic assays in the absence of HER2/neu expression

• Signaling pathway analysis:

  • Use SPSB1 antibodies in combination with phospho-specific antibodies to key signaling molecules

  • Investigate how SPSB1 modulates signaling cascades like the c-MET pathway

  • Examine how SPSB1 affects the balance between pro-survival and pro-apoptotic signals

• In vivo tumor regression and recurrence models:

  • Implement orthotopic tumor models with SPSB1 modulation

  • Track tumor regression and recurrence after therapy

  • Use SPSB1 antibodies for immunohistochemical analysis of tumor sections

Research has shown that SPSB1-expressing BT474 cells treated with Lapatinib exhibited lower caspase-3/7 activity and less cell death than control cells, while knockdown of SPSB1 in Hs578T cells increased apoptosis following treatment with chemotherapeutic agents .

What are the considerations when using SPSB1 antibodies to study its interaction with NF-κB pathway components?

When investigating SPSB1's interactions with NF-κB pathway components, particularly p65, researchers should consider several methodological aspects:

• Stimulus-dependent interactions:

  • SPSB1's interaction with p65 is enhanced after IL-1β treatment

  • Design co-immunoprecipitation experiments with appropriate stimulation time points

  • Include both stimulated and unstimulated conditions (e.g., IL-1β treatment for 30 minutes has been shown to enhance this interaction)

• Domain-specific interactions:

  • Use domain deletion mutants like SPSB1-ΔSOCS to map interaction regions

  • Both full-length SPSB1 and SPSB1-ΔSOCS interact with p65, indicating the SOCS-box domain is dispensable for binding to p65

  • Employ tagged constructs to facilitate immunoprecipitation studies

• Subcellular localization considerations:

  • SPSB1 shows prominent nuclear localization after IL-1β treatment

  • Use subcellular fractionation followed by immunoblotting

  • Combine with immunofluorescence to visualize co-localization with p65

• Functional consequence analysis:

  • Assess transcriptional consequences using reporter assays

  • Examine expression of NF-κB target genes like iNOS and IL-6

  • Compare wild-type SPSB1 with domain mutants to link physical interactions with functional outcomes

Previous research has shown that SPSB1 depletion resulted in significantly higher expression of both iNOS and IL-6 (150 and 660 fold, respectively) compared to control cells after IL-1β treatment .

What are the optimal storage and handling conditions for SPSB1 antibodies?

Based on commercial antibody information, SPSB1 antibodies should be stored at -20°C where they remain stable for one year after shipment . The antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

For optimal handling:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Keep on ice during experiment setup

• Centrifuge briefly before opening the vial to collect liquid at the bottom

• When diluting, use buffers recommended by the manufacturer

Despite manufacturer information suggesting aliquoting is unnecessary for -20°C storage , dividing antibodies into single-use aliquots is generally considered best practice to maintain antibody performance over time.

How can researchers optimize SPSB1 antibody dilutions for different experimental applications?

Optimal dilution of SPSB1 antibodies is sample-dependent and should be determined empirically for each experimental system . Based on research practices, the following optimization approaches are recommended:

• For Western blotting:

  • Begin with a dilution range test (1:500, 1:1000, 1:2000, 1:5000)

  • Include positive controls (cells known to express SPSB1) and negative controls (SPSB1 knockdown samples)

  • Evaluate signal-to-noise ratio at each dilution

  • Select the dilution that provides clear specific bands with minimal background

• For immunofluorescence:

  • Start with manufacturer recommendations or 1:100-1:500 dilution range

  • Include antigen retrieval optimization if working with fixed tissues

  • Test blocking conditions to minimize non-specific binding

  • Verify specificity using SPSB1 overexpression and knockdown controls

• For immunoprecipitation:

  • Typically requires higher antibody concentrations (2-5 μg per IP reaction)

  • Optimize lysis buffer conditions to preserve protein-protein interactions

  • Consider crosslinking the antibody to beads to prevent antibody contamination in the eluate

• For ELISA applications:

  • Perform checkerboard titration with different antibody and antigen concentrations

  • Determine optimal coating concentration if using SPSB1 antibody as capture antibody

  • Test different blocking agents to minimize background

The optimization process should incorporate proper controls at each step and may need to be repeated when switching to new antibody lots or sample types.

How can SPSB1 antibodies be used to study viral infection responses?

SPSB1 has been shown to limit innate immune responses to viral infections such as RSV. SPSB1 suppresses the expression of IFN-β and IFN-dependent genes like ISG54 and OAS1 during viral infection . Researchers can use SPSB1 antibodies to investigate these mechanisms through:

• Viral infection models:

  • Infect cells stably expressing GFP or SPSB1 with viruses (e.g., RSV at 2 PFU/cell)

  • After 24 hours, assess innate immune response markers

  • Use SPSB1 antibodies to confirm expression levels in experimental systems

  • Previous research showed that SPSB1 expression reduced the levels of IFN-β, ISG54, and OAS1 mRNA in RSV-infected cells

• Signaling pathway analysis:

  • Use SPSB1 antibodies along with antibodies against phosphorylated signaling molecules

  • Investigate how SPSB1 affects pathways like RIG-I and TLR signaling

  • Examine potential interactions with viral sensing adaptors

• SPSB1 localization during infection:

  • Perform immunofluorescence to track SPSB1 localization changes during viral infection

  • Co-stain with markers of viral replication and innate immune signaling components

• Chromatin immunoprecipitation (ChIP):

  • Use SPSB1 antibodies for ChIP to investigate whether SPSB1 associates with promoters of innate immune genes

  • Combine with p65 ChIP to understand how SPSB1 affects NF-κB binding to target promoters

These approaches can help elucidate how SPSB1 modulates antiviral responses and potentially identify novel therapeutic targets for enhancing immunity or controlling excessive inflammation.

How do SPSB1 antibodies help in distinguishing between different SPSB family members?

The SPSB protein family includes multiple members (SPSB1, SPSB2, SPSB3, and SPSB4) that share structural similarities but may have distinct functions. When using antibodies to study SPSB1 specifically, researchers should consider:

• Epitope selection:

  • Choose antibodies targeting epitopes unique to SPSB1

  • Avoid antibodies targeting the conserved SOCS box domain, which could cross-react with other family members

  • Perform sequence alignment of SPSB family members to identify divergent regions

• Validation with recombinant proteins:

  • Test antibody specificity against recombinant SPSB1, SPSB2, SPSB3, and SPSB4

  • Use western blotting to confirm selective recognition of SPSB1

  • Include size controls as SPSB family members have different molecular weights

• Genetic approaches:

  • Validate antibody specificity in cells with CRISPR-mediated knockout of SPSB1

  • Test antibody reactivity in cells overexpressing individual SPSB family members

  • Use siRNA knockdown of specific SPSB proteins to confirm antibody specificity

• Mass spectrometry verification:

  • Perform immunoprecipitation with SPSB1 antibody followed by mass spectrometry

  • Analyze peptide coverage to confirm specific enrichment of SPSB1 rather than other family members

By employing these approaches, researchers can ensure their observations are specific to SPSB1 and not confounded by cross-reactivity with other SPSB family members, which is crucial for accurate interpretation of experimental results.

What are common issues when using SPSB1 antibodies and how can they be resolved?

When working with SPSB1 antibodies, researchers may encounter several common challenges. Based on research experiences with similar proteins, here are potential issues and solutions:

• Weak or absent signal in Western blot:

  • Increase antibody concentration or incubation time

  • Enhance protein loading (SPSB1 may be expressed at low levels in some cells)

  • Try different detection methods with increased sensitivity

  • Verify SPSB1 expression in your cell type (cells like Hs578T express high levels, while MDA-MB-231 and BT474 express lower levels)

  • Check sample preparation (ensure protease inhibitors are included in lysis buffer)

• High background in immunofluorescence:

  • Optimize blocking conditions (try BSA, normal serum, or commercial blockers)

  • Increase washing steps duration and number

  • Reduce primary antibody concentration

  • Test different fixation methods (paraformaldehyde vs. methanol)

  • Use antigen retrieval methods if appropriate

• Non-specific bands in Western blot:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Include competitive blocking with immunizing peptide if available

  • Use gradient gels to better separate proteins of similar molecular weight

  • Include positive controls (SPSB1 overexpression) and negative controls (SPSB1 knockdown)

• Inconsistent co-immunoprecipitation results:

  • Remember that SPSB1 interactions may be stimulus-dependent (e.g., enhanced p65 interaction after IL-1β treatment)

  • Optimize lysis conditions to preserve protein-protein interactions

  • Consider crosslinking approaches for transient interactions

  • Increase cell number or protein concentration for low-abundance interactions

By systematically addressing these issues, researchers can improve the reliability and consistency of their experiments with SPSB1 antibodies.