SPTBN1 Antibody

What is SPTBN1 and what is its primary function in cellular systems?

SPTBN1 is the non-erythrocytic form of β-spectrin (also called β-fodrin), a member of the superfamily of F-actin cross-linking proteins. It forms micrometer-scale networks associated with plasma membranes and functions as a scaffolding protein for protein sorting, cell adhesion, and migration . SPTBN1 is ubiquitously expressed but particularly abundant in the brain, where it is essential for neuronal development and connectivity . The protein plays a critical role in central nervous system development and interacts with calmodulin in a calcium-dependent manner, suggesting involvement in calcium-dependent movement of the cytoskeleton at the membrane .

What are the optimal applications for SPTBN1 antibodies in research?

Based on validated research applications, SPTBN1 antibodies are primarily used for:

The choice of application should be guided by the specific research question and the validated reactivity of the selected antibody .

What are the recommended dilutions for SPTBN1 antibodies in different applications?

Optimal dilutions vary significantly between antibodies and applications:

It is essential to titrate each antibody in your specific experimental system to determine optimal working dilutions, as these can be sample-dependent . For heat-mediated antigen retrieval in IHC applications, TE buffer pH 9.0 is generally recommended, with citrate buffer pH 6.0 as an alternative .

How does SPTBN1 expression correlate with cancer prognosis?

SPTBN1 demonstrates context-dependent prognostic significance across different cancer types:

• Tumor suppressor role: Reduced expression correlates with shorter survival in hepatocellular cancer, pancreatic cancer, and other gastrointestinal malignancies .

• Kidney renal clear cell carcinoma (KIRC): Higher SPTBN1 expression associates with better prognosis. Multivariate analysis confirms SPTBN1 as an independent predictive factor (HR = 0.647, 95% CI = 0.489–0.854, P = 0.002) . ROC curve analysis shows high diagnostic value (AUC: 0.692; 95% CI = 0.644–0.741) .

• Uveal melanoma (UVM): Contrary to KIRC, higher SPTBN1 expression correlates with worse outcomes .

SPTBN1 expression is frequently lower in cancer tissues compared to adjacent non-tumor tissues in pan-cancer analysis . This differential expression pattern makes SPTBN1 a potentially valuable prognostic biomarker, particularly when incorporated into nomograms with other clinical parameters .

What is the mechanism of SPTBN1 in neurodevelopmental disorders?

SPTBN1 plays a critical role in neuronal development, with pathogenic variants causing a distinct neurodevelopmental syndrome:

• Clinical presentation: Global developmental delays, language and motor impairments, mild to severe intellectual disability, autistic features, seizures, behavioral abnormalities, hypotonia, and variable dysmorphic facial features .

• Molecular mechanisms: Pathogenic variants lead to:

  • Loss-of-function effects affecting protein stability

  • Gain-of-function effects creating novel protein interactions

  • Dominant negative effects disrupting normal protein function

• Structural consequences: CH domain variants result in depletion of membrane-bound βII-spectrin and formation of cytosolic aggregates containing actin and αII-spectrin. These variants disrupt cytoskeletal organization and neuronal development .

The 28 unique SPTBN1 variants identified in affected individuals include 17 classified as pathogenic, 9 as likely pathogenic, and 2 as variants of uncertain significance (VUS) . Mouse models with brain βII-spectrin deficiency recapitulate the developmental and behavioral phenotypes observed in affected individuals .

How does SPTBN1 interact with the tumor immune microenvironment?

SPTBN1 demonstrates cancer type-specific associations with immune infiltration:

• Kidney renal carcinoma (KIRC): Significant negative correlations between SPTBN1 expression and pro-tumor immune cells (Treg cells, Th2 cells, monocytes, M2-macrophages) and immune modulatory genes (e.g., TNFSF9) .

• Uveal melanoma (UVM): Opposite pattern to KIRC, with positive correlations between SPTBN1 and immunosuppressive cells .

These findings suggest SPTBN1 functions as a dual marker for predicting cancer prognosis and response to immunotherapy, potentially influencing immunotherapy efficacy in KIRC patients and anti-cancer targeted treatments in UVM patients . The ESTIMATE algorithm can be used to analyze correlations between SPTBN1 expression and stromal/immune components in the tumor microenvironment .

What molecular mechanisms mediate SPTBN1's regulation of GPT2 in cancer?

SPTBN1 regulates GPT2 (Glutamic-Pyruvic Transaminase 2) at the post-transcriptional level through a direct RNA-binding mechanism:

• mRNA stability regulation: SPTBN1 functions as an RNA-binding protein that binds to the 3'-UTR region of GPT2 mRNA, reducing its stability .

• Experimental validation:

  • Actinomycin D stability assays show SPTBN1-depleted cells have more stable GPT2 mRNAs

  • RNA immunoprecipitation (RIP) demonstrates direct binding of SPTBN1 to GPT2

  • Dual-luciferase reporter assays confirm SPTBN1 reduces wild-type GPT2 3'-UTR activity but not mutated versions

• Expression correlation: Significant negative correlation between SPTBN1 and GPT2 expression in multiple datasets:

  • TCGA-KIRC database: Pearson: R = −0.176, P < 0.001; Spearman: R = −0.203, P < 0.001

  • NJMU ccRCC cohort: Pearson: R = −0.703, P < 0.001; Spearman: R = −0.668, P < 0.001

  • TMA immunohistochemical data: Pearson: R = −0.274, P < 0.001; Spearman: R = −0.292, P < 0.001

This regulatory mechanism contributes to SPTBN1's tumor-suppressive role in clear cell renal cell carcinoma by modulating GPT2-dependent metabolic pathways .

What are the technical challenges in detecting SPTBN1 in experimental settings?

Several technical considerations should be addressed when working with SPTBN1 antibodies:

• Protein size: SPTBN1 is a large protein (calculated molecular weight of 275 kDa), which can present challenges for efficient transfer in Western blots and extraction from tissues .

• Epitope selection: Different antibodies target different epitopes (e.g., AA 2100-2200, AA 2096-2256), requiring careful selection based on the region of interest and experimental design .

• Antigen retrieval: For IHC applications, proper antigen retrieval is critical. TE buffer pH 9.0 is generally recommended, with citrate buffer pH 6.0 as an alternative .

• Cross-reactivity: When studying SPTBN1 across species, verify the cross-reactivity of the antibody with your species of interest. Available antibodies have been validated for human, mouse, rat, rabbit, and pig samples .

• Variant analysis: When studying SPTBN1 variants, complementary functional assays may be needed to determine pathogenicity, as demonstrated in studies of neurodevelopmental variants .

How can researchers validate the specificity of SPTBN1 antibodies?

A multi-step validation approach is recommended:

• Positive controls: Use tissues/cells known to express SPTBN1 such as:

  • Brain tissue (mouse, rat, human)

  • Kidney tissue (human, mouse)

  • Cell lines: HEK-293, HeLa, Jurkat

• Molecular weight verification: Confirm detection at the expected molecular weight (275 kDa observed)

• Multiple application testing: Validate across multiple applications (WB, IHC, IF) to ensure consistent results

• Knockdown/knockout controls: When possible, use SPTBN1 knockdown or knockout models as negative controls

• Antibody KD determination: Consider KD values (equilibrium dissociation constant) when available, as recombinant antibodies typically show 1-2 orders of magnitude higher affinity than mouse monoclonal antibodies

What is the optimal protocol for detecting SPTBN1 in tissue microarrays for cancer prognosis studies?

Based on successful implementations in cancer research:

This methodology has successfully identified SPTBN1 as a prognostic marker in KIRC and other cancers, demonstrating its utility in clinical oncology research .