SEC61G Antibody

What is SEC61G and what is its role in cellular function?

SEC61G functions as a critical subunit of the SEC61 translocon complex, which is essential for translocation of newly synthesized proteins across the endoplasmic reticulum membrane. As part of this complex, SEC61G contributes to the structural stability of the translocon and facilitates proper protein processing, including glycosylation. The SEC61 complex, composed of SEC61α/β/γ (SEC61A/B/G) heterotrimer, regulates almost all secreted and transmembrane proteins entering the ER . Recent studies have revealed that SEC61G plays a crucial role in the glycosylation, stabilization, and membrane presentation of immune checkpoint ligands (ICLs), which has significant implications for immune regulation and tumor biology .

SEC61G has been identified as frequently co-amplified with EGFR in glioblastoma, where it promotes the glycosylation and stabilization of immune checkpoint ligands including PD-L1, PVR, and PD-L2 . These processes inhibit antitumor CD8+ T cell activity, contributing to immune evasion and tumor progression. Pancancer analysis has shown that SEC61G gene amplification is common in many tumor types, with GBM showing the highest frequency (~36%) .

How does SEC61G interact with the SEC61 translocon complex?

The intact SEC61 complex is crucial for the translocation of nascent polypeptides into the ER lumen, where they undergo essential modifications including folding, glycosylation, and quality control before being transported to their final destinations within the cell . Disruption of SEC61G function can significantly impact protein processing, particularly for membrane-bound proteins like immune checkpoint ligands that require proper glycosylation for their stability and function .

What are the optimal applications for SEC61G antibodies in research?

SEC61G antibodies can be employed in multiple research applications:

• Western blotting: For detection and quantification of SEC61G protein levels in cell or tissue lysates. When working with SEC61G (a small protein of approximately 7-8 kDa), use high percentage gels (12-15%) and optimize transfer conditions for small proteins.

• Immunohistochemistry (IHC): To visualize SEC61G expression patterns in tissue sections. Research has shown negative correlation between SEC61G protein levels and CD8+ T cell infiltration in human GBM tissues .

• Immunofluorescence (IF): To examine subcellular localization of SEC61G in cells, typically showing ER membrane distribution. Double staining with ER markers like GRP94 can confirm proper localization .

• Immunoprecipitation (IP): To isolate SEC61G and its interacting partners. Studies have demonstrated that SEC61G interacts with immune checkpoint ligands including PD-L1, PVR, and PD-L2 through reciprocal immunoprecipitation assays .

• Flow cytometry: For analyzing SEC61G expression at the single-cell level.

These applications enable researchers to investigate SEC61G's expression patterns, interactions, and functional roles in normal physiology and disease states, particularly in cancer research where SEC61G has been implicated in tumor progression and immune evasion .

How is SEC61G implicated in glioblastoma pathogenesis?

SEC61G has significant implications in glioblastoma biology. Research has revealed several key aspects of its involvement:

Immune evasion mechanism: SEC61G promotes immune evasion by enhancing the glycosylation, stabilization, and membrane presentation of immune checkpoint ligands (ICLs) including PD-L1, PVR, and PD-L2 . This process inhibits anti-tumor immune responses, particularly CD8+ T cell activity. Gene Set Enrichment Analysis (GSEA) of TCGA-GBM datasets showed that SEC61G levels negatively correlate with populations of total T cells and activated CD8+ T cells .

Therapeutic implications: Depletion of SEC61G in experimental models significantly inhibits tumor growth in immunocompetent mice but has only a marginal effect in immunodeficient nude mice, indicating critical involvement of T cell-mediated immune surveillance in tumor suppression . SEC61G depletion significantly prolongs survival in mouse models and increases the population of cytotoxic CD8+ T cells .

What is the relationship between SEC61G and EGFR amplification?

SEC61G and EGFR share a close genomic and functional relationship in glioblastoma:

Co-amplification: The SEC61G gene is located at chromosome 7p11, in close proximity to the EGFR gene, and is frequently co-amplified with EGFR in GBM tumors . This co-amplification results in elevated expression of both genes, contributing to aggressive tumor behavior.

Independent functions: While SEC61G can moderately regulate EGFR levels and affect the EGFR pathway in GBM cells, its effects on immune evasion (particularly PD-L1 expression) appear to be largely independent of EGFR signaling . Experiments showed that while SEC61G depletion moderately down-regulates EGFR levels and represses the EGFR pathway in GBM cells, EGF treatment only marginally reversed the effect of SEC61G depletion on PD-L1 expression, indicating that SEC61G induces PD-L1 expression mostly independent of EGFR .

Therapeutic resistance: The co-amplification of SEC61G with EGFR may partially explain the inefficacy of EGFR-targeted therapies alone in GBM . The authors suggest that SEC61G-mediated immune evasion may act as a critical mechanism for tumorigenesis in EGFR-amplified tumors, making SEC61G an important additional target for combination therapy approaches .

How can SEC61G antibodies be used to study immune evasion mechanisms?

SEC61G antibodies serve as valuable tools for investigating the mechanisms by which SEC61G contributes to immune evasion in cancer:

Protein interaction studies: Immunoprecipitation with SEC61G antibodies can help identify its binding partners in the protein translocation and glycosylation machinery. Reciprocal immunoprecipitation assays using SEC61G antibodies have demonstrated that SEC61G interacts with immune checkpoint ligands including PD-L1, PVR, and PD-L2 .

Co-localization studies: Using SEC61G antibodies in combination with antibodies against immune checkpoint ligands allows visualization of their cellular co-localization. Double staining with antibodies against PD-L1 and GRP94 (an ER marker) has shown that depletion of SEC61G inhibits translocation of PD-L1 into the ER .

Protein expression analysis: SEC61G antibodies enable researchers to assess SEC61G expression levels in different tumor types and correlate them with immune cell infiltration markers. In human GBM tissues, the protein levels of SEC61G were found to negatively correlate with the infiltration and cytolytic activity of CD8+ T cells .

Tumor microenvironment analysis: Immunohistochemistry with SEC61G antibodies on tumor sections can reveal spatial relationships between SEC61G-expressing tumor cells and infiltrating immune cells, providing insights into the immune microenvironment .

By employing SEC61G antibodies in these applications, researchers can decipher how SEC61G promotes glycosylation and stabilization of immune checkpoint ligands, contributing to immune evasion and tumor progression .

What are the key protocols for studying SEC61G-mediated protein glycosylation?

SEC61G plays a crucial role in protein glycosylation, particularly for immune checkpoint ligands. The following protocols are essential for studying SEC61G-mediated glycosylation:

Glycosylation detection by Western blotting:

• Prepare cell lysates in RIPA buffer with protease inhibitors

• Separate proteins on 7-12% SDS-PAGE gels

• Transfer to PVDF membranes

• Block and probe with antibodies against glycosylated proteins of interest (e.g., PD-L1)

• Compare SEC61G-depleted cells with controls to observe changes in glycosylation patterns

Research has demonstrated that depletion of SEC61G significantly decreases the levels of glycosylated forms of PD-L1 and correspondingly increases non-glycosylated PD-L1 in GBM cells .

Glycosidase treatment assays:

• Treat cell lysates with glycosidases like PNGase F (removes all N-linked glycans) or Endoglycosidase H (removes only high-mannose glycans)

• Analyze mobility shifts by Western blotting

• Compare glycosylation patterns between SEC61G-depleted and control cells

Pulse-chase experiments for glycoprotein maturation:

• Pulse cells with radioactive precursors or amino acids

• Chase with non-radioactive medium

• Harvest cells at various timepoints

• Immunoprecipitate proteins of interest

• Analyze glycosylation status over time using SEC61G antibodies

ER translocation visualization:

• Perform immunofluorescence staining with antibodies against SEC61G and glycoproteins of interest

• Co-stain with ER markers (e.g., GRP94)

• Use confocal microscopy to visualize co-localization

• Compare translocation efficiency in SEC61G-manipulated cells

Studies have shown that depletion of SEC61G inhibits translocation of PD-L1 into the ER, as demonstrated by double staining with antibodies against PD-L1 and GRP94 .

How can researchers validate SEC61G antibody specificity?

Comprehensive validation of SEC61G antibody specificity is crucial for reliable experimental results:

Genetic validation:

• siRNA/shRNA knockdown: Demonstrate reduced antibody signal after SEC61G knockdown. Studies have shown successful depletion of SEC61G using shRNAs in various cell models .

• CRISPR/Cas9 knockout: Show complete loss of signal in SEC61G knockout cells

• Overexpression: Confirm increased signal in cells overexpressing SEC61G

Biochemical validation:

• Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding

• Western blotting: Confirm single band of expected molecular weight (~7-8 kDa)

• Multiple antibodies: Use antibodies recognizing different epitopes and confirm consistent results

Application-specific validation:

• For IHC/IF: Show appropriate subcellular localization (ER membrane pattern)

• For IP: Demonstrate enrichment of SEC61G in immunoprecipitated fraction and confirm interaction with known binding partners such as other SEC61 complex components

• For flow cytometry: Compare with isotype controls and validate with knockdown controls

Documentation and controls:

• Maintain detailed records of all validation experiments

• Include appropriate positive and negative controls in all experiments

• Always run parallel experiments with isotype control antibodies

A properly validated SEC61G antibody should demonstrate clear signal reduction upon SEC61G depletion, appropriate subcellular localization at the ER membrane (possibly co-localizing with established ER markers like GRP94), and specific immunoprecipitation of SEC61G protein .

What approaches can be used to study SEC61G-protein interactions?

Several complementary approaches can be employed to study SEC61G interactions with other proteins:

Co-immunoprecipitation (Co-IP):

• Lyse cells in mild detergent buffers (e.g., 1% NP-40 or 0.5% CHAPS) to preserve protein interactions

• Incubate lysates with SEC61G antibodies coupled to beads

• Wash and elute bound proteins

• Analyze by Western blotting or mass spectrometry

Reciprocal immunoprecipitation assays in GBM cells have successfully demonstrated that SEC61G interacts with immune checkpoint ligands including PD-L1, PVR, and PD-L2 .

Proximity labeling methods:

• BioID: Fuse biotin ligase to SEC61G to biotinylate proximal proteins

• APEX2: Use peroxidase-mediated labeling to identify proteins in close proximity to SEC61G

• These methods are particularly useful for detecting transient or weak interactions

Crosslinking approaches:

• Treat intact cells with membrane-permeable crosslinkers

• Lyse cells and immunoprecipitate SEC61G

• Reverse crosslinks and identify interacting proteins

• This approach can capture transient interactions that might be lost during conventional IP

Microscopy-based methods:

• Immunofluorescence co-localization: Double staining with SEC61G antibodies and antibodies against potential interacting partners

• Förster resonance energy transfer (FRET): For detecting close proximity between fluorescently labeled proteins

• Proximity ligation assay (PLA): Visualize and quantify protein interactions in situ

Research has demonstrated effective co-localization studies using double staining with antibodies against SEC61G, PD-L1, and ER markers like GRP94 to visualize their interactions and subcellular localization .

How can SEC61G inhibition be used in combination with other cancer treatments?

SEC61G inhibition shows promising potential for combination with other cancer treatments, particularly in glioblastoma:

Combination with EGFR inhibitors:
Studies have demonstrated that SEC61 inhibition with Eeyarestatin I (ES I) in combination with the EGFR inhibitor erlotinib almost completely abrogated tumor formation in experimental models . While erlotinib alone had only a moderate effect on tumor growth, the combination therapy significantly inhibited tumor growth and promoted the infiltration of antitumor CD8+ T cells .

Protocol for combination therapy assessment:

• Establish appropriate tumor models (subcutaneous or orthotopic)

• Administer SEC61 inhibitor (e.g., ES I) alone, EGFR inhibitor alone, or in combination

• Monitor tumor growth and survival endpoints

• Analyze immune cell infiltration and activation by flow cytometry and immunohistochemistry

• Examine expression of immune checkpoint ligands and their glycosylation status

Mechanistic basis for combination:
The efficacy of this combination lies in targeting two complementary mechanisms. EGFR inhibitors block oncogenic signaling pathways, while SEC61G inhibition prevents immune evasion by reducing the glycosylation and membrane presentation of immune checkpoint ligands . Treatment of GBM cells with ES I has been shown to decrease the levels of PD-L1, PVR, and PD-L2 in a dose-dependent manner, decrease glycosylated PD-L1, and increase PD-L1 ubiquitination .

Potential for immunotherapy combinations:
Given SEC61G's role in immune checkpoint ligand expression, combining SEC61G inhibition with immune checkpoint blockade (e.g., anti-PD-1/PD-L1 therapy) represents another promising strategy. By reducing the expression of multiple immune checkpoint ligands simultaneously, SEC61G inhibition could enhance the efficacy of targeted immunotherapies .

How does SEC61G affect immune checkpoint ligand glycosylation and stability?

SEC61G plays a critical role in regulating immune checkpoint ligand glycosylation and stability through several mechanisms:

Glycosylation pathway:
SEC61G facilitates the translocation of newly synthesized immune checkpoint ligands (ICLs) into the ER, where they undergo N-linked glycosylation . When SEC61G is depleted, this translocation is impaired, leading to decreased glycosylation of ICLs including PD-L1, PVR, and PD-L2 . Experiments have shown that depletion of SEC61G significantly decreases the levels of the glycosylated form of PD-L1 and correspondingly increases the levels of non-glycosylated PD-L1 in GBM cells .

Protein stability regulation:
Glycosylation protects ICLs from degradation, and SEC61G-mediated glycosylation inhibits their ubiquitination . Research has demonstrated that SEC61G depletion promotes PD-L1 ubiquitination, leading to increased proteasomal degradation . Similarly, treatment with the SEC61 inhibitor Eeyarestatin I (ES I) increases PD-L1 ubiquitination .

Membrane localization:
SEC61G is required for proper membrane presentation of ICLs. Studies have shown that SEC61G depletion significantly decreases both the total levels and membrane-bound forms of PD-L1, PVR, and PD-L2 in GBM cells . These effects were confirmed using multiple techniques including flow cytometry and immunostaining .

Interaction with ICLs:
SEC61G directly interacts with ICLs as demonstrated by reciprocal immunoprecipitation assays . This interaction is likely crucial for their proper processing through the secretory pathway. The direct binding suggests that SEC61G may serve as a chaperone or quality control factor for these proteins during their maturation .

What is the impact of SEC61G on antitumor immunity in in vivo models?

SEC61G has profound effects on antitumor immunity in experimental models:

Tumor growth inhibition:
Depletion of SEC61G strongly inhibits tumor growth in immunocompetent mice but has only a marginal effect in immunodeficient nude mice, indicating a critical involvement of T cell-mediated immune surveillance in tumor suppression . Studies using intracranial implantation of GL-26 cells expressing SEC61G shRNAs demonstrated significantly prolonged survival in immunocompetent mice (medium survival duration of 20 days in control vs. 42 days in shSEC61G groups) .

Enhanced CD8+ T cell infiltration and activation:
SEC61G depletion significantly increases the population of CD8+ T cells (CD8+CD3+) and induces cytotoxic CD8+ T cell activation (GZMB+CD8+) in tumor tissues . This was confirmed by both flow cytometry analysis and immunostaining of mouse tissues demonstrating enhanced CD8α and GZMB double staining in SEC61G-depleted tumors .

Reduced immune checkpoint ligand expression:
Immunostaining of mouse tissues demonstrated that SEC61G depletion inhibits the expression of PD-L1, a critical immune checkpoint molecule expressed in tumor cells . This reduction in immune checkpoint ligands likely contributes to enhanced T cell activity against tumor cells.

Dependency on CD8+ T cells:
The antitumor effects of SEC61G depletion are dependent on CD8+ T cells, as administration of an anti-CD8α monoclonal antibody significantly reversed the effect of SEC61G depletion on tumor growth and reduced the survival of GBM-bearing mice (42 days in shSEC61G plus IgG2b vs. 24 days in shSEC61G plus CD8α mAb) . This confirms that SEC61G depletion inhibits GBM tumorigenesis primarily by promoting CD8+ T cell infiltration and cytotoxic activity .

How do SEC61 inhibitors affect protein translocation and glycosylation?

SEC61 inhibitors, such as Eeyarestatin I (ES I), have specific effects on protein translocation and glycosylation:

Inhibition of protein translocation:
ES I inhibits SEC61-mediated protein translocation at the ER . As a SEC61 translocon inhibitor, it prevents the entry of newly synthesized proteins into the ER lumen, disrupting the first critical step in the secretory pathway. This affects all proteins that rely on the SEC61 complex for their entry into the ER, including secreted and membrane proteins .

Reduced glycosylation of immune checkpoint ligands:
Treatment of GBM cells with ES I decreases the levels of immune checkpoint ligands including PD-L1, PVR, and PD-L2 in a dose-dependent manner . Furthermore, ES I specifically decreases the level of glycosylated PD-L1 in GBM cells , demonstrating its impact on the post-translational modification process.

Enhanced protein ubiquitination and degradation:
By preventing proper glycosylation, ES I promotes ubiquitination of proteins like PD-L1 . Research has shown that ES I treatment results in increased PD-L1 ubiquitination, which leads to proteasomal degradation and reduced protein levels .

Antitumor effects:
ES I substantially inhibits tumor growth in experimental models . Moreover, ES I in combination with the EGFR inhibitor erlotinib almost completely prevents tumor formation . In mouse tumor tissues, erlotinib in combination with ES I significantly promotes the infiltration of antitumor CD8+ T cells , indicating enhanced immune surveillance.

Therapeutic applications:
ES I has been reported to induce cell death, suppress tumor growth, and sensitize tumors to tyrosine kinase inhibitors (TKIs) in various cancers . The ability of SEC61 inhibitors to target both protein expression and immune evasion mechanisms makes them particularly promising for combination therapy approaches .

What are the emerging therapeutic strategies targeting SEC61G in cancer?

Several emerging therapeutic strategies targeting SEC61G show promise for cancer treatment:

Direct SEC61G inhibition:
SEC61 inhibitors like Eeyarestatin I (ES I) have demonstrated significant antitumor activity in preclinical models . These compounds inhibit SEC61-mediated protein translocation at the ER, preventing glycosylation and membrane presentation of immune checkpoint ligands . Studies have shown that ES I substantially inhibits tumor growth in mouse models .

Combination with EGFR inhibitors:
Given the frequent co-amplification of SEC61G with EGFR in glioblastoma, combining SEC61G inhibitors with EGFR tyrosine kinase inhibitors (TKIs) represents a promising strategy . Research has demonstrated that while erlotinib (an EGFR inhibitor) alone had only moderate effects on tumor growth, ES I in combination with erlotinib almost completely prevented tumor formation in experimental models . This synergistic effect occurs because SEC61G inhibition addresses a key resistance mechanism in EGFR-amplified tumors .

Enhancement of immunotherapy:
By reducing the expression of multiple immune checkpoint ligands (PD-L1, PVR, PD-L2), SEC61G inhibition may enhance the efficacy of immune checkpoint blockade therapies . The ability of SEC61G inhibition to increase CD8+ T cell infiltration and activation in tumors provides a strong rationale for combining SEC61G targeting with immunotherapies .

Clinical development:
While not specifically mentioned in the search results for SEC61G, related SEC61 inhibitors are advancing in clinical development. For example, KZR-261, a SEC61 inhibitor, is currently in a phase I clinical trial for patients with advanced solid tumors . This suggests growing interest in targeting the SEC61 complex as a therapeutic strategy in cancer.

What are common technical challenges when working with SEC61G antibodies?

Researchers may encounter several technical challenges when working with SEC61G antibodies:

Detection of low molecular weight protein:
SEC61G is a small protein (~7-8 kDa), which can present challenges for detection. For Western blotting, use high percentage gels (15-20%) to properly resolve small proteins, and optimize transfer conditions (consider PVDF membranes and wet transfer at low voltage for extended time). Ensure appropriate molecular weight markers that include low molecular weight range are used for accurate size determination.

Membrane protein solubilization:
As a component of the SEC61 translocon complex, SEC61G is an integral membrane protein that requires appropriate detergents for solubilization. Use membrane-specific lysis buffers containing effective detergents like NP-40, Triton X-100, or CHAPS at optimized concentrations. Avoid excessive heating of samples, as membrane proteins can aggregate at high temperatures.

Complex stability preservation:
The SEC61 complex (SEC61α/β/γ) functions as a heterotrimer, and disruption of the complex may affect antibody recognition. Consider native conditions for certain applications to maintain complex integrity. When studying SEC61G interactions with other proteins (like immune checkpoint ligands), gentle lysis conditions are essential to preserve these associations .

Subcellular localization visualization:
SEC61G localizes to the ER membrane, which requires proper fixation and permeabilization for immunofluorescence studies. Optimize fixation protocols (typically 4% paraformaldehyde) and permeabilization conditions (0.1-0.3% Triton X-100) for clear visualization of ER patterns. Co-staining with established ER markers like GRP94 can help confirm proper localization .

Signal specificity verification:
Ensuring signal specificity is crucial, particularly when studying a protein involved in complex cellular processes. Always validate antibody specificity using SEC61G knockdown or knockout controls. Consider using multiple antibodies targeting different epitopes to confirm results and include appropriate negative controls in all experiments.

How can researchers optimize SEC61G detection in different experimental systems?

Optimization strategies for SEC61G detection across different experimental systems:

Western blotting optimization:

• Sample preparation: Use RIPA or NP-40 lysis buffers with protease inhibitors

• Gel selection: 15-20% polyacrylamide gels for optimal resolution of small proteins

• Transfer conditions: Wet transfer at low voltage (25V) for extended time (2 hours) onto PVDF membranes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody dilution: Titrate primary antibody (typically 1:500-1:1000) for optimal signal-to-noise ratio

• Enhanced chemiluminescence detection: Use high-sensitivity ECL substrates for detecting low-abundance proteins

• Loading controls: Include appropriate controls for normalization (β-actin, GAPDH, or other housekeeping proteins)

Immunofluorescence optimization:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1-0.3% Triton X-100 for 5-10 minutes

• Blocking: 1-5% BSA or normal serum for 30-60 minutes

• Antibody incubation: Overnight at 4°C for primary antibody (1:100-1:500 dilution)

• Co-staining: Include ER markers like GRP94 to confirm proper localization

• Mounting: Use anti-fade mounting medium to prevent photobleaching

• Imaging: Confocal microscopy for detailed subcellular localization

Immunohistochemistry considerations:

• Antigen retrieval: Critical for FFPE tissues; try different methods (citrate pH 6.0 or EDTA pH 9.0)

• Background reduction: Use appropriate blocking of endogenous peroxidase and biotin

• Signal amplification: Consider tyramide signal amplification for low-abundance proteins

• Counterstaining: Hematoxylin for nuclear visualization

• Controls: Include known positive tissues and negative controls

Flow cytometry parameters:

• Cell preparation: Gentle dissociation methods to preserve membrane integrity

• Fixation/permeabilization: Optimize for intracellular detection (commercially available kits)

• Antibody titration: Determine optimal concentration using serial dilutions

• Controls: Include isotype controls and SEC61G-depleted samples

• Gating strategy: Define positive populations based on appropriate controls

How can contradictory results regarding SEC61G function be reconciled?

When facing contradictory results regarding SEC61G function, consider the following approaches:

Methodological differences assessment:
Carefully evaluate differences in experimental approaches, including:

• Detection methods: Different antibodies or assays may yield varying results

• Cell systems: SEC61G function may vary between cell types or patient-derived samples

• Experimental conditions: Acute (siRNA) versus chronic (stable knockdown) SEC61G modulation may have different outcomes

• Analysis techniques: Variations in quantification or normalization methods can lead to discrepancies

Biological context consideration:
SEC61G's functions may be context-dependent:

• Genetic background: Different model systems may have varying compensatory mechanisms

• Disease heterogeneity: SEC61G's role may differ across GBM subtypes or patient populations

• Immune status: Effects on immune evasion will be more evident in immunocompetent models than in immune-deficient systems

• Protein expression levels: Threshold effects may exist where partial depletion has different consequences than complete knockout

Experimental validation approaches:
To resolve contradictions, implement rigorous validation:

• Independent approaches: Use multiple techniques to study the same process (e.g., both biochemical assays and imaging for glycosylation analysis)

• Genetic complementation: Rescue experiments with SEC61G re-expression to confirm specificity

• Dose-response relationships: Examine effects across a range of SEC61G expression levels

• Time-course analyses: Distinguish between immediate and adaptive responses to SEC61G modulation

Reconciliation example:
In cases where contradictory results exist regarding SEC61G's impact on immune checkpoint ligand expression, consider that:

• SEC61G may affect different immune checkpoint ligands to varying degrees

• Both direct (glycosylation, stabilization) and indirect (signaling pathway) effects may be involved

• The impact on immune responses will be observable in immunocompetent models but might be missed in immune-deficient systems

• Effects on EGFR-dependent and EGFR-independent pathways might vary between experimental systems