GOLM1 Antibody

What is GOLM1 and what cellular functions does it perform?

GOLM1 is a type II transmembrane protein originally located in the Golgi apparatus that cycles among membranous compartments, including sorting endosomes and the plasma membrane . It serves as a specific cargo adaptor mediating transport processes between the trans-Golgi network and plasma membrane, playing a crucial role in protein processing and trafficking . GOLM1 has been identified as a promoter of proliferation, invasion, and migration in various human malignancies, including hepatocellular carcinoma, prostate cancer, esophageal cancer, gastric cancer, and cutaneous melanoma .

What are the standard applications for GOLM1 antibodies in cancer research?

GOLM1 antibodies are typically employed in multiple experimental techniques:

• Western blotting (WB): Used at dilutions of 1:500-1:5000 to detect GOLM1 protein expression in cell lysates and tissue samples

• Immunohistochemistry (IHC): Applied to evaluate GOLM1 expression patterns in tumor tissues and correlate with clinical outcomes

• Co-immunoprecipitation (Co-IP): Utilized to study protein-protein interactions between GOLM1 and other signaling molecules

• Flow cytometry (FC): Employed to detect cellular GOLM1 expression levels

• ELISA: Used to quantify GOLM1 protein levels in biological samples

How is GOLM1 expression correlated with cancer progression?

GOLM1 expression has shown significant correlations with cancer progression across multiple tumor types:

What are the recommended protocols for validating GOLM1 antibody specificity?

To ensure antibody specificity, researchers should implement a multi-layered validation approach:

• Positive and negative controls: Use cell lines with known high GOLM1 expression (e.g., MHCC-97H, HCC-LM3) as positive controls and low-expressing lines (e.g., PLC, Hep3B) as negative controls

• Knockdown validation: Confirm antibody specificity by detecting decreased signal in GOLM1-knockdown cells. Multiple GOLM1-specific shRNAs can be employed to validate knockdown efficiency

• Recombinant protein controls: Use purified recombinant GOLM1 protein as a positive control in immunoblotting experiments

• Multiple antibody comparison: Compare results using different antibodies targeting distinct GOLM1 epitopes to confirm detection consistency

• Cross-reactivity testing: Evaluate potential cross-reactivity with related Golgi proteins to ensure specificity

How should researchers design GOLM1 knockdown experiments to study its function?

For effective GOLM1 knockdown studies:

• Multiple shRNA constructs: Generate at least 3-4 GOLM1-specific shRNAs to control for off-target effects. Select the construct with the most significant knockdown efficiency, as demonstrated in multiple studies

• Rescue experiments: Reintroduce recombinant GOLM1 that is not sensitive to the shRNA (shRES-GOLM1) to rescue the phenotype and exclude off-target effects

• Validation methods: Confirm knockdown efficiency at both mRNA (RT-qPCR) and protein (Western blot) levels

• Controls: Include non-target shRNA controls (shNT) in all experiments

• Functional assays: Assess changes in cell proliferation, migration, invasion, and immune regulation upon GOLM1 knockdown

What in vivo models are most appropriate for studying GOLM1's role in cancer progression?

Several in vivo models have been successfully used to study GOLM1 function:

• Subcutaneous xenograft models: Implanting GOLM1-manipulated cancer cells subcutaneously in immunodeficient mice to assess tumor growth, as demonstrated with PC9 cells

• Orthotopic models: More physiologically relevant models involving implantation of cancer cells into the organ of origin

• Syngeneic models in immunocompetent mice: Essential for studying immune-related functions of GOLM1, as shown with H22 hepatoma and MCA205 fibrosarcoma cells in C57BL/6 mice

• GOLM1 knockout models: Generation of GOLM1-knockout cancer cell lines using CRISPR/Cas9 for implantation in mice

• Imaging approaches: Incorporate FDG-PET/CT imaging to accurately evaluate tumor growth dynamics in vivo

How does GOLM1 modulate immune responses in the tumor microenvironment?

GOLM1 influences immune responses through multiple mechanisms:

• PD-L1 regulation: GOLM1 promotes CSN5-mediated deubiquitination and stabilization of PD-L1 in HCC cells, enhancing immune checkpoint signaling

• Exosomal PD-L1 transport: GOLM1 increases exosomal PD-L1 levels and facilitates its transfer to tumor-associated macrophages (TAMs), contributing to immune suppression

• Macrophage polarization: GOLM1 expression correlates with increased infiltration of immunosuppressive TAMs in the tumor microenvironment

• T cell suppression: High GOLM1 expression is associated with increased expression of T cell suppression markers (PD-1, TIM-3) and decreased effector cytokines (IFN-γ, GZMB) in CD8+ T cells

• Autophagy suppression: GOLM1 inhibits autophagy-mediated anti-tumor immunity through AKT/mTOR pathway regulation and affects extracellular ATP release

What methodologies are effective for studying GOLM1's interaction with immune checkpoint proteins?

Researchers can employ several approaches to study GOLM1's interactions with immune checkpoint proteins:

• Co-immunoprecipitation (Co-IP): Used to verify interactions between GOLM1 and immune-related proteins like PD-L1 and CSN5

• GST pulldown assays: Helpful in determining direct protein-protein interactions and mapping interaction domains, as demonstrated with GOLM1 and PD-L1

• Proximity ligation assays: Can detect protein-protein interactions in situ in fixed cells or tissues

• Flow cytometry: Effective for quantifying surface expression of immune checkpoint proteins in different cell populations

• Immunofluorescence co-localization: Used to visualize subcellular co-localization of GOLM1 with immune checkpoint proteins

• In vitro T cell function assays: Measure T cell suppression through co-culture systems with GOLM1-manipulated cancer cells

How can researchers evaluate GOLM1-mediated effects on T cell function?

To assess GOLM1's impact on T cell function, researchers should consider:

• Flow cytometry analysis: Measure expression of T cell suppression markers (PD-1, TIM-3) and activation markers (IFN-γ, GZMB) in tumor-infiltrating CD8+ T cells from GOLM1-high versus GOLM1-low tumors

• T cell proliferation assays: Assess T cell proliferation capacity using CFSE dilution or Ki67 staining when exposed to conditioned media from GOLM1-manipulated cancer cells

• Cytotoxicity assays: Evaluate cytolytic activity of T cells against GOLM1-high versus GOLM1-low cancer cells

• Cytokine profiling: Measure cytokine production (IFN-γ, TNF-α, IL-2) by T cells in response to GOLM1-manipulated cancer cells

• Immune checkpoint blockade response: Compare the efficacy of anti-PD-1/PD-L1 therapy in GOLM1-high versus GOLM1-low tumors in vivo

What mechanisms underlie GOLM1's regulation of PD-L1 expression and transport?

GOLM1 regulates PD-L1 through several distinct mechanisms:

• Post-translational stabilization: GOLM1 promotes COP9 signalosome 5 (CSN5)-mediated deubiquitination of PD-L1, increasing its stability in cancer cells

• Protein-protein interaction: GOLM1 directly interacts with PD-L1 through its region spanning residues 36-205, as demonstrated by GST pulldown assays

• Exosomal packaging: GOLM1 increases the transport of PD-L1 into exosomes, potentially by suppressing Rab27b expression

• Exosome secretion: GOLM1 associates with exosome markers (CD63, CD9, TSG101, Alix) to facilitate exosome production and release

• Intercellular transfer: Exosomes derived from GOLM1-high cancer cells can transfer PD-L1 to macrophages, increasing PD-L1 expression on TAMs

How does GOLM1 influence cancer cell signaling pathways?

GOLM1 affects multiple signaling pathways in cancer cells:

• AKT/mTOR pathway: GOLM1 regulates AKT/mTOR signaling to affect autophagy formation and extracellular ATP release

• P53 signaling: In lung cancer, GOLM1 overexpression enhances P53 phosphorylation at site S315 but inhibits the formation of P53 tetramers

• EGFR/RTK recycling: GOLM1 modulates EGFR/RTK cell-surface recycling to drive hepatocellular carcinoma metastasis

• Cell cycle regulation: GOLM1 affects cell cycle progression, with GOLM1 knockdown decreasing the percentage of cells in G2 phase

• Cytoskeletal rearrangement: GOLM1 overexpression increases actin polymerization, potentially contributing to enhanced cell motility

How can researchers study GOLM1's role in exosome-mediated intercellular communication?

To investigate GOLM1's role in exosome-mediated communication:

• Exosome isolation: Use differential ultracentrifugation, size exclusion chromatography, or commercial kits to isolate exosomes from conditioned media of GOLM1-manipulated cells

• Exosome characterization: Verify exosome identity through nanoparticle tracking analysis, transmission electron microscopy, and detection of exosomal markers (CD63, CD9, TSG101)

• Protein cargo analysis: Use Western blotting or mass spectrometry to analyze protein content (including PD-L1) in exosomes derived from GOLM1-high versus GOLM1-low cells

• Functional assays: Study the effects of purified exosomes on recipient cells (e.g., macrophages) by measuring changes in phenotype, function, and protein expression

• Co-culture systems: Use transwell co-culture systems to study intercellular communication between GOLM1-manipulated cancer cells and immune cells

• In vivo tracking: Label exosomes with fluorescent dyes or membrane reporters to track their biodistribution in vivo

How should researchers interpret contradictory findings regarding GOLM1's role across different cancer types?

When facing contradictory findings:

• Context-dependent functions: Recognize that GOLM1 may have tissue-specific and context-dependent functions across different cancer types

• Methodological differences: Evaluate variations in experimental approaches, including antibody specificity, knockdown efficiency, and model systems used

• Comprehensive pathway analysis: Employ systems biology approaches to map GOLM1's interactions with different cellular pathways in specific contexts

• Single-cell analysis: Consider heterogeneity within tumor samples that might explain seemingly contradictory population-level findings

• Integration of multiomics data: Combine transcriptomic, proteomic, and functional data to build a more comprehensive understanding of GOLM1's role

What approaches are most effective for correlating GOLM1 expression with immune cell infiltration?

For robust correlation analyses:

• Multiplex immunohistochemistry: Simultaneously evaluate GOLM1 expression and immune cell markers (CD8, CD68, PD-L1) in tissue sections

• Flow cytometry immunophenotyping: Quantify various immune cell populations in relation to GOLM1 expression levels

• Spatial transcriptomics: Map GOLM1 expression and immune cell distribution with spatial resolution in tissue sections

• Single-cell RNA sequencing: Profile tumor and immune cell populations to correlate GOLM1 expression with specific immune cell states

• Computational deconvolution: Use algorithms like CIBERSORT or xCell to estimate immune cell fractions from bulk RNA-seq data of GOLM1-high versus GOLM1-low tumors

• Correlation statistics: Apply appropriate statistical tests (Pearson, Spearman) and multivariate analyses to evaluate associations between GOLM1 expression and immune parameters

How can researchers distinguish between GOLM1's direct effects and secondary consequences in signaling pathways?

To differentiate direct from indirect effects:

• Timing experiments: Perform time-course analyses to identify immediate versus delayed responses following GOLM1 manipulation

• Acute induction systems: Use inducible expression systems (e.g., Tet-On) to study immediate consequences of GOLM1 activation

• Direct interaction studies: Employ co-IP, GST pulldown, and proximity ligation assays to identify direct protein binding partners of GOLM1

• Domain mapping: Generate truncated GOLM1 constructs to identify functional domains responsible for specific interactions and effects

• Pathway inhibitors: Use specific inhibitors targeting suspected downstream pathways to determine dependence on these pathways

• Rescue experiments: Test whether direct introduction of purported downstream effectors can rescue phenotypes observed in GOLM1-depleted cells