SPOCK1 Antibody

What is SPOCK1 and why is it significant in research?

SPOCK1 is a proteoglycan of approximately 49-54 kDa that plays important roles in cell-cell and cell-matrix interactions . It may contribute to various neuronal mechanisms in the central nervous system under normal physiological conditions . SPOCK1 has gained significant research interest due to its roles in multiple cancers, including lung adenocarcinoma, prostate cancer, and hepatocellular carcinoma, where its upregulation is frequently associated with poor prognosis . The protein was first discovered in 1992, isolated from seminal plasma, and then cloned from brain tissue in 1997 . Understanding SPOCK1's functions and expression patterns is valuable for exploring cancer progression mechanisms and identifying potential therapeutic targets.

What are the primary applications of SPOCK1 antibodies in research?

SPOCK1 antibodies are primarily used in Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P) . These applications allow researchers to:

• Detect and quantify SPOCK1 protein expression in tissue samples and cell lysates

• Examine subcellular localization of SPOCK1 in different cell types

• Compare SPOCK1 expression between normal and diseased tissues

• Investigate correlations between SPOCK1 expression and clinicopathological features

• Study the effects of experimental manipulations on SPOCK1 expression

For example, SPOCK1 antibodies have been used to demonstrate that this protein is highly expressed in tumor cells compared to normal cells, and that it colocalizes with mitochondrial markers in hepatoma cell lines .

What is the typical molecular weight of SPOCK1 and what band pattern should researchers expect?

The predicted molecular weight of SPOCK1 is approximately 49-50 kDa, but researchers typically observe a band at around 50 kDa on Western blots . This slight difference between predicted and observed molecular weight may be attributed to post-translational modifications of the protein. When using commercial antibodies such as ab229935, researchers can expect to see a single distinct band at approximately 50 kDa in human samples such as 293T whole cell lysate, as well as in mouse brain tissue lysate . It's important to note that glycosylation patterns may vary between tissue types, potentially resulting in slight variations in the observed molecular weight.

What are the recommended protocols for using SPOCK1 antibodies in Western blotting?

For optimal Western blotting results with SPOCK1 antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Use whole cell lysates or tissue homogenates with complete protease inhibitors.

• Loading amount: 20-30 μg of total protein per lane is typically sufficient.

• Recommended dilution: For antibodies like ab229935, a dilution of 1:2000 has been validated .

• Secondary antibody: Use a compatible secondary antibody (e.g., goat polyclonal to rabbit IgG) at approximately 1:50000 dilution .

• Expected results: Look for a band at approximately 50 kDa.

• Controls: Include positive controls such as brain tissue lysates (mouse or human) or 293T cell lysate .

When troubleshooting, consider that SPOCK1 detection may require optimization of lysis buffers to ensure complete solubilization of the proteoglycan components.

How should researchers optimize SPOCK1 antibodies for immunohistochemistry?

For effective immunohistochemical detection of SPOCK1:

• Fixation: Standard formalin fixation and paraffin embedding is compatible with SPOCK1 detection.

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended.

• Blocking: Use 5-10% normal serum from the same species as the secondary antibody.

• Primary antibody incubation: A 1:100 to 1:500 dilution range is appropriate for most SPOCK1 antibodies in IHC-P applications.

• Detection system: Both chromogenic (DAB) and fluorescent detection methods are suitable.

• Counterstaining: Hematoxylin works well for visualizing tissue architecture alongside SPOCK1 staining.

When interpreting results, researchers should note that SPOCK1 shows varying expression patterns across tissues. In normal liver, SPOCK1 exhibits modest cytoplasmic positivity in hepatocytes, with stronger expression in cirrhotic hepatocytes and variable intensity in cancer cells . Endothelial cells typically display intense cytoplasmic positivity, while connective tissue is generally negative .

What controls should be used when working with SPOCK1 antibodies?

Appropriate controls are essential for validating SPOCK1 antibody specificity and ensuring reliable results:

• Positive tissue controls:

  • Brain tissue (normal expression)

  • Lung adenocarcinoma tissue (overexpression)

  • Prostate cancer tissue (variable expression based on Gleason grade)

  • Hepatocellular carcinoma samples (increased expression compared to normal liver)

• Negative controls:

  • Primary antibody omission

  • Isotype-matched irrelevant antibody

  • Tissues known to have minimal SPOCK1 expression

• Technical validation controls:

  • SPOCK1 knockdown or knockout cell lines

  • Recombinant SPOCK1 protein for antibody pre-absorption

  • Parallel testing with multiple SPOCK1 antibodies targeting different epitopes

In experimental hepatocarcinogenesis models, researchers observed that SPOCK1 is barely detectable in healthy mouse livers but becomes increasingly expressed in transformed hepatocytes, providing a useful system for antibody validation .

How does SPOCK1 subcellular localization inform its function?

Though traditionally considered an extracellular matrix proteoglycan, SPOCK1 exhibits interesting subcellular localization patterns that suggest additional functions:

• Cytoplasmic localization: SPOCK1 concentration increases in the cytoplasm of hepatocytes during progression from normal cells to cirrhotic liver and hepatocellular carcinoma .

• Mitochondrial association: In hepatoma cell lines, cytoplasmic SPOCK1 colocalizes with mitochondrial markers such as MitoTracker and TOMM20 (a protein in the outer mitochondrial membrane) . This unexpected localization suggests potential roles in mitochondrial function or signaling.

• Nuclear presence: SPOCK1 can also be detected in the cell nucleus in certain contexts .

These diverse localization patterns suggest that SPOCK1 may have currently unidentified intracellular functions beyond its characterized extracellular roles. Researchers investigating SPOCK1 should consider multiple subcellular compartments when designing experiments and interpreting results.

What signaling pathways are modulated by SPOCK1 and how can researchers study them?

SPOCK1 influences multiple signaling pathways relevant to cancer progression:

SPOCK1 downregulation in hepatoma cell lines inhibits cell proliferation, upregulates p21 and p27, and interferes with pAkt and CDK4 expression . A tyrosine kinase array revealed that inhibition of SPOCK1 in liver cancer cells altered MAPK signaling and downregulated several members of the Src family, which relate to cancer aggressiveness .

When studying these pathways, researchers should consider using:

• SPOCK1 knockdown via siRNA or CRISPR-Cas9

• SPOCK1 overexpression systems

• Pathway-specific inhibitors to determine epistatic relationships

• Phospho-protein arrays to identify affected signaling nodes

How does SPOCK1 expression correlate with immune infiltration in the tumor microenvironment?

SPOCK1 expression is associated with specific patterns of immune cell infiltration in tumors:

• Positive correlation with immunosuppressive components:

  • Cancer-associated fibroblasts

  • Myeloid-derived suppressor cells

  • Tumor-associated macrophages

  • Regulatory T cells

• Negative correlation with anti-tumor immunity:

  • CD8+ T-cell infiltration is negatively correlated with SPOCK1 expression in lung adenocarcinoma

• Relationship with immune checkpoint molecules:

  • SPOCK1 expression positively correlates with PD-L1 at both mRNA and protein levels

  • May play a regulatory role in modulating the immunosuppressive microenvironment through PD-L1

• Association with specific tumor microenvironment features:

  • Angiogenesis

  • Fibroblast activity

  • Pro-tumorigenic immune infiltration

  • EMT proliferation score

These findings suggest that SPOCK1 may influence tumor immune evasion mechanisms and could affect response to immunotherapies. Researchers interested in this aspect should consider multiplex immunohistochemistry, flow cytometry, or single-cell RNA sequencing to further characterize the relationship between SPOCK1 expression and immune cell populations.

How can SPOCK1 expression patterns be used for cancer prognosis and treatment planning?

SPOCK1 expression has demonstrated prognostic value in multiple cancer types:

In prostate cancer specifically, high SPOCK1 expression correlates with:

• Advanced stage disease (p = 0.018)

• Advanced tumor (T) value (p = 0.014)

• Higher Gleason grade groups, particularly groups 3 and 4 (p = 0.044 and 0.003)

For translational applications, researchers could develop SPOCK1 expression assessment as part of prognostic panels for cancer patients. Standardized immunohistochemical scoring systems would need to be established and validated across multiple cohorts.

What is the relationship between SPOCK1 and therapeutic resistance in cancer?

SPOCK1 has been implicated in resistance to several cancer therapies:

• Targeted therapy resistance:

  • SPOCK1 is significantly overexpressed in osimertinib-resistant lung cancer cells

  • Deletion of SPOCK1 inhibits proliferation of osimertinib-resistant cells and resensitizes them to the drug

  • SPOCK1 may serve as a marker for predicting osimertinib resistance

• Radiotherapy resistance:

  • SPOCK1 may contribute to radioresistance through its relationship with PD-L1

  • PD-L1 induces radioresistance by stimulating cell migration and promoting epithelial-mesenchymal transition

  • Radioresistance can potentially be reversed by blocking PD-L1 expression

• Blood-brain barrier considerations:

  • SPOCK1 contributes to blood-brain barrier (BBB) formation and maintenance

  • Downregulation of SPOCK1 may temporarily open the BBB to facilitate entry of therapeutic agents

  • This could improve efficacy of chemotherapy, targeted therapy, and immunotherapy for brain metastases

Researchers investigating therapeutic resistance should consider SPOCK1 as a potential therapeutic target, particularly in combination treatment strategies.

What potential exists for targeting SPOCK1 in cancer therapy?

Based on current research, several approaches for therapeutic targeting of SPOCK1 show promise:

• Direct inhibition strategies:

  • Development of specific SPOCK1 inhibitors (currently in preclinical stages)

  • Downregulation via RNA interference approaches

  • Targeting SPOCK1 to increase drug penetration across the blood-brain barrier

• Combinatorial approaches:

  • Using SPOCK1 inhibition to sensitize resistant cells to osimertinib or other targeted therapies

  • Combining SPOCK1 targeting with radiotherapy to overcome radioresistance

  • Sequential therapy with radiation and immune checkpoint inhibitors

• Monitoring effectiveness:

  • Using SPOCK1 expression as a biomarker for treatment response

  • Developing correlative analyses between SPOCK1 expression and drug sensitivity profiles

Future directions in this field include clinical trials of SPOCK1 inhibitors for treating brain metastases from non-small cell lung cancer (NSCLC) and other malignancies. Additionally, correlation analysis between target drugs and SPOCK1 gene expression may identify relatively sensitive drugs for treating NSCLC brain metastasis patients .