CSMD3 Antibody

What is CSMD3 and why is it important in research?

CSMD3 is a large transmembrane protein composed of 3707 amino acid residues, encoded by a gene located on human chromosome 8q23.3-q24.1 . It contains multiple CUB and Sushi domains and functions as a potential tumor suppressor protein with roles in immune regulation . CSMD3 has gained significant research interest due to its dysregulation in various cancer types and its expression in neuronal tissues . Research utilizing CSMD3 antibodies has revealed its importance in understanding tumor microenvironments, cancer progression mechanisms, and neurological disorders, making it a valuable target for both oncology and neuroscience investigations .

What are the primary applications for CSMD3 antibodies in research?

CSMD3 antibodies are valuable tools primarily used in Western blot and ELISA applications for detecting and analyzing CSMD3 protein expression in various cell types and tissues . In immunohistochemistry/immunofluorescence studies, these antibodies enable visualization of CSMD3's cellular localization, particularly in neuronal cells (co-localized with NeuN) and oligodendrocyte cells (co-localized with MBP) . They are also essential for investigating CSMD3's role in cancer development, immune regulation, and neurological function through co-immunoprecipitation experiments to identify protein-protein interactions, and for validating gene knockout models such as CSMD3-/- mice . The multiple applications of these antibodies facilitate comprehensive analysis of CSMD3's diverse biological functions.

What cell types and tissues express CSMD3?

Based on immunofluorescence studies using CSMD3 antibodies, CSMD3 protein is predominantly expressed in neuronal tissues, with significant expression detected in hippocampal neurons (co-localized with NeuN) and cerebellar tissues (co-localized with calbindin) . Additionally, CSMD3 expression has been observed in oligodendrocyte-like cells (MO3.13 cells) where it co-localizes with myelin basic protein (MBP) . In cancer research, CSMD3 expression has been extensively studied in ovarian cancer tissues, where its mutation status correlates with immune cell infiltration patterns . CSMD3 has also been detected in liver cells, including the normal liver cell line LO2 and hepatocellular carcinoma cell line HepG2, which serve as positive controls for antibody validation .

How do CSMD3 mutations correlate with tumor mutation burden and immune infiltration?

Comprehensive analysis of whole-exome sequencing data from 410 American and 93 British ovarian cancer patients revealed that CSMD3 mutations significantly correlate with elevated tumor mutation burden (TMB) . This correlation is particularly noteworthy as TMB is a recognized biomarker for immunotherapy response. Gene Set Enrichment Analysis (GSEA) demonstrated that ovarian cancer samples harboring CSMD3 mutations show significant enrichment in immune-related pathways, including PPAR signaling, graft versus host disease, intestinal immune network for IgA production, primary immunodeficiency, allograft rejection, and systemic lupus erythematosus .

CIBERSORT algorithm analysis revealed distinct immune cell infiltration patterns in CSMD3-mutated versus wild-type tumors, with significantly higher CD8+ T cell infiltration and lower M0 macrophage presence in CSMD3-mutated samples . These findings suggest CSMD3 mutation status may serve as a potential biomarker for predicting immunotherapy response and influence the tumor immune microenvironment through modulation of specific immune cell populations.

What are the implications of CSMD3 knockout models for neurological research?

CSMD3 knockout models (CSMD3-/- mice) generated through CRISPR/Cas9-mediated gene targeting have revealed critical insights into CSMD3's neurological functions . These models demonstrate that CSMD3 deficiency leads to significant motor impairments and autism-like behaviors, establishing CSMD3 as an important protein in neurological development and function . Immunostaining of brain tissues from these models using CSMD3 antibodies alongside neuronal markers (NeuN), Purkinje cell markers (calbindin), interneuron markers (parvalbumin/PV, calretinin/CR), oligodendrocyte markers (MBP), astrocyte markers (GFAP), and microglial markers (Iba1) has enabled detailed characterization of CSMD3's expression pattern in various neural cell types and its absence in knockout models .

The comparison between wild-type and CSMD3-/- mice provides valuable insights into how CSMD3 deficiency affects brain development, neural circuit formation, and behavioral outcomes, making these models essential for understanding CSMD3's role in neurological disorders and potential therapeutic approaches.

How can CSMD3 mutation status be leveraged as a biomarker in cancer research?

For translational research, CSMD3 mutation serves as a potential predictive biomarker for immunotherapy response due to its association with altered immune infiltration patterns, particularly increased CD8+ T cell presence . Researchers can utilize CSMD3 antibodies to validate mutation-specific effects on protein expression and localization, correlating these findings with genomic data. Additionally, the enrichment of immune-related pathways in CSMD3-mutated tumors suggests this gene's involvement in modulating the tumor immune microenvironment, offering avenues for developing combination therapy approaches targeting both CSMD3 and immune checkpoint pathways.

What are the optimal protocols for CSMD3 immunostaining in different neural tissues?

For optimal CSMD3 immunostaining in neural tissues, researchers should follow tissue-specific protocols that have been validated in published studies. For cerebellar tissue sections, the recommended protocol involves perfusion with 4% paraformaldehyde (PFA), post-fixation for 8 hours, and cryoprotection in 30% sucrose overnight at 4°C . Sagittal sections (30 μm thickness) should be cut using a freezing microtome, blocked with 5% donkey serum for 1 hour, and incubated with rabbit anti-CSMD3 antibody (1:300 dilution) alongside cell-type-specific markers .

For cultured hippocampal neurons, a modified protocol using 2% PFA fixation for 30 minutes, permeabilization with 0.1% Triton X-100, and blocking with 5% donkey serum for 40 minutes is recommended . Primary antibody incubation should be conducted overnight at 4°C using rabbit anti-CSMD3 (1:300) and mouse anti-NeuN (1:200) . For both protocols, appropriate secondary antibodies include Alexa Fluor 488 donkey anti-rabbit (1:1000) and Cy3 donkey anti-mouse (1:1000), with DAPI (100 ng/ml) counterstaining for nuclear visualization . These optimized protocols ensure specific signal detection while minimizing background fluorescence.

What controls should be included when validating CSMD3 antibody specificity?

Comprehensive validation of CSMD3 antibody specificity requires multiple controls to ensure reliable experimental results. Primary controls should include CSMD3 knockout models (CSMD3-/- mice tissues or CRISPR/Cas9-edited cell lines) as negative controls to confirm antibody specificity . This genetic ablation approach provides the most stringent control for antibody validation. Positive control samples should include tissues or cell lines with confirmed CSMD3 expression, such as LO2 and HepG2 cells as indicated in antibody specifications .

Technical controls should include: (1) primary antibody omission control to assess secondary antibody non-specific binding; (2) isotype control using non-immune IgG at equivalent concentration to evaluate background staining; (3) peptide/antigen pre-absorption test using the immunogen sequence (amino acids 220-440 of human CSMD3) to confirm epitope-specific binding ; and (4) cross-reactivity testing in multiple species if cross-species applications are planned. Additionally, orthogonal validation using alternative detection methods (Western blot, ELISA, immunoprecipitation) with the same antibody reinforces confidence in specificity. This multi-tiered validation approach ensures experimental rigor when using CSMD3 antibodies for research applications.

What are the methodological considerations for analyzing CSMD3 in cancer tissue samples?

Analyzing CSMD3 in cancer tissue samples requires careful methodological considerations across multiple analytical platforms. For immunohistochemical or immunofluorescence analysis, tissue fixation conditions must be optimized as overfixation can mask CSMD3 epitopes. Antigen retrieval methods should be carefully selected based on the specific CSMD3 antibody requirements—typically, heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended .

When correlating CSMD3 protein expression with mutation status, researchers should implement parallel genomic analysis through targeted sequencing or whole-exome sequencing of the same samples. For tumor microenvironment studies, multiplex immunofluorescence staining that combines CSMD3 antibody with immune cell markers (CD8, CD4, macrophage markers) enables spatial relationship analysis between CSMD3-expressing cells and tumor-infiltrating immune cells . Additionally, tissue microarrays containing multiple patient samples can facilitate high-throughput screening of CSMD3 expression patterns across cancer subtypes.

For quantitative analysis, digital pathology platforms with appropriate algorithms for membrane protein quantification should be employed, with careful distinction between specific membrane staining and background signal. Finally, researchers should consider tumor heterogeneity by analyzing multiple regions within the same tumor to account for spatial variations in CSMD3 expression.

How should discrepancies between CSMD3 genomic data and protein expression be reconciled?

Post-transcriptional and post-translational regulatory mechanisms should be examined through parallel analysis of CSMD3 mRNA (RT-qPCR) and protein levels (Western blot). Nonsense-mediated decay may eliminate mutant CSMD3 transcripts with premature stop codons, while certain missense mutations might affect protein stability rather than expression . For complex cases, protein degradation assays using proteasome inhibitors can help determine if mutation-induced protein instability explains the discrepancy.

In cancer samples specifically, tumor heterogeneity must be considered by microdissection of different tumor regions for comparative analysis. Finally, single-cell analysis techniques can resolve cell-type-specific differences in CSMD3 expression that might be masked in bulk tissue analysis. By systematically addressing these factors, researchers can better interpret seeming contradictions between genomic and protein-level data.

What approaches can resolve contradictory findings regarding CSMD3's role in cancer progression?

Researchers should implement mechanistic studies using CRISPR/Cas9-mediated CSMD3 knockout or overexpression in relevant cancer cell lines, followed by comprehensive phenotypic assays (proliferation, migration, invasion) and immune co-culture experiments to directly assess CSMD3's functional impact. Cancer-type specificity should be thoroughly evaluated, as CSMD3 may have divergent roles across different malignancies.

The apparent contradictions might be explained by CSMD3's dual roles in different biological processes. Using phospho-proteomics and interactome analysis with co-immunoprecipitation followed by mass spectrometry can identify context-dependent CSMD3 binding partners and affected signaling pathways. Additionally, patient stratification based on CSMD3 mutation type (not just presence/absence) and location might resolve some contradictions, as different mutations may have distinct functional consequences.

Finally, in vivo models with conditional CSMD3 modification at different cancer stages can help determine whether CSMD3's role shifts during tumor evolution, potentially explaining apparently contradictory findings in the literature.

What factors should be considered when interpreting CSMD3 immunostaining patterns in neuronal tissues?

Interpreting CSMD3 immunostaining patterns in neuronal tissues requires consideration of multiple technical and biological factors. As a membrane protein with multiple domains, CSMD3's subcellular localization is complex—researchers should carefully distinguish between cell surface expression and intracellular vesicular patterns, using confocal microscopy with Z-stack imaging for accurate spatial resolution .

Developmental timing significantly impacts CSMD3 expression patterns, as CSMD3 mRNA has been detected in both embryonic and postnatal brain tissues . Age-matched control tissues are therefore essential for comparative studies. The neuron-specific expression of CSMD3 should be validated using co-localization with multiple neuronal markers (NeuN for general neurons, calbindin for Purkinje cells) to accurately characterize cell-type specificity .

Brain region heterogeneity must be considered when comparing CSMD3 expression across different neuroanatomical structures. The established protocol for cerebellar tissue requires specific conditions (sagittal 30-μm-thick serial sections) that may differ from optimal conditions for other brain regions . Additionally, post-mortem interval and fixation conditions can significantly affect CSMD3 epitope preservation and detection sensitivity in brain tissues.

Finally, when studying neurological disease models, researchers should distinguish between altered CSMD3 expression as a cause versus consequence of pathological changes by implementing time-course analyses and correlating CSMD3 patterns with functional and behavioral outcomes.