ATG4C Antibody

What is ATG4C and why is it important in autophagy research?

ATG4C (Autophagy Related 4C Cysteine Peptidase) is a member of the autophagin protein family and plays a key role in autophagy by mediating both proteolytic activation and delipidation of ATG8 family proteins . It functions as a cysteine protease that cleaves the C-terminal amino acid of ATG8 proteins MAP1LC3 and GABARAPL2, revealing a C-terminal glycine that is essential for their conjugation to phosphatidylethanolamine (PE) and insertion into membranes .

ATG4C is particularly important in research because:

• It contributes to autophagy regulation, which is essential for cell homeostasis and remodeling during differentiation, metamorphosis, non-apoptotic cell death, and aging

• It has distinct functional properties compared to other ATG4 family members, showing weaker proteolytic activation but stronger delipidation activity than ATG4B

• Reduced levels of autophagy have been associated with malignant tumors, suggesting ATG4C may play a role in controlling unregulated cell growth linked to cancer

Understanding ATG4C protein characteristics is crucial for selecting appropriate antibodies:

• Molecular Weight: ATG4C has a calculated molecular weight of approximately 52 kDa, typically observed at 52-55 kDa on Western blots

• Protein Structure: ATG4C consists of 458 amino acids

• Expression Pattern: Highly expressed in skeletal muscle, heart, liver, and testis

• Subcellular Localization: Primarily cytoplasmic

• Species Conservation: ATG4C sequence shows conservation across human, mouse, and rat, though cross-reactivity varies by antibody

A band of unknown origin at approximately 23 kDa may be detected with some antibodies, with the intensity of this band reduced under certain conditions .

How does ATG4C function differ from other ATG4 family members, and how can antibodies help distinguish them?

ATG4C exhibits distinct functional characteristics compared to other ATG4 family members, particularly ATG4B:

• Functional Differences: Compared to ATG4B (the major protein for proteolytic activation of ATG8 proteins), ATG4C shows weaker ability to cleave the C-terminal amino acid of ATG8 proteins but displays stronger delipidation activity

• Autophagy Roles: Unlike other family members, ATG4C is weakly or not involved in phagophore growth during mitophagy

• Stress Response: ATG4C-deficient mice display tissue-specific decreases in LC3 lipidation primarily under stressful conditions such as prolonged starvation

• Tumor Suppression: Atg4C-deficient mice exhibit increased susceptibility to chemical carcinogen-induced fibrosarcomas, suggesting ATG4C may contribute to tumor suppression

To distinguish between ATG4 family members, researchers should:

• Use highly specific antibodies that recognize unique epitopes

• Validate specificity using knockout cell lines (e.g., the Human ATG4C knockout HEK-293T cell line shows loss of signal with specific ATG4C antibodies)

• Compare expression patterns across tissues, as ATG4C shows highest expression in skeletal muscle, heart, liver, and testis

What are the best validation methods to confirm ATG4C antibody specificity?

Rigorous validation of ATG4C antibodies is essential for reliable research outcomes:

Recommended Validation Methods:

• Genetic Knockdown/Knockout Controls:

  • Use ATG4C knockout cell lines (e.g., Human ATG4C knockout HEK-293T cell line) to confirm antibody specificity

  • Compare wild-type and ATG4C siRNA-treated samples to verify signal reduction

• Multiple Antibody Validation:

  • Use antibodies targeting different epitopes (N-terminal, C-terminal, and internal regions) to confirm consistent detection

  • Compare monoclonal (e.g., [2E10H7]) and polyclonal antibodies targeting ATG4C

• Cross-Reactivity Assessment:

  • Test antibody reactivity across multiple species if cross-species studies are planned

  • Verify minimal cross-reactivity with other ATG4 family members (ATG4A, ATG4B, ATG4D)

• Application-Specific Validation:

  • For Western blotting: Confirm band size (expected 52-55 kDa)

  • For IHC/IF: Include peptide competition assays to confirm signal specificity

  • For IP: Validate pulled-down protein by mass spectrometry

• Loading Controls and References:

  • Include appropriate loading controls (e.g., GAPDH) when performing Western blotting

  • Use tissues known to express high levels of ATG4C (skeletal muscle, heart, liver, testis) as positive controls

How can ATG4C antibodies be used to investigate the relationship between autophagy and cancer development?

ATG4C has been implicated in cancer development, with ATG4C-deficient mice showing increased susceptibility to chemical carcinogen-induced fibrosarcomas . Researchers can use ATG4C antibodies to investigate this relationship through several approaches:

Methodological Approaches:

• Tumor Tissue Analysis:

  • Compare ATG4C expression levels in tumor versus normal tissues using IHC and Western blotting

  • Correlate ATG4C expression with tumor stage, grade, and patient outcomes

  • Examine co-localization with other autophagy markers in tumor sections

• Functional Studies in Cancer Models:

  • Monitor ATG4C expression and activity during carcinogenesis using antibodies in combination with autophagy flux assays

  • Track changes in ATG4C's dual roles (proteolytic activation and delipidation) during cancer progression

  • Examine ATG4C's interaction with other autophagy proteins in cancer cells using co-immunoprecipitation with specific antibodies

• Stress Response Analysis:

  • Investigate ATG4C expression under various stressors relevant to the tumor microenvironment (hypoxia, nutrient deprivation)

  • Compare autophagy flux between normal and cancer cells under stress conditions using ATG4C antibodies alongside other autophagy markers

• Therapeutic Response Monitoring:

  • Assess changes in ATG4C expression and localization in response to anticancer therapies, particularly those affecting autophagy

  • Use ATG4C antibodies to monitor autophagy modulation as a potential biomarker for treatment response

What is the optimal protocol for ATG4C detection by Western blotting?

Based on published methodology and technical information, here is an optimized protocol for ATG4C detection by Western blotting:

Sample Preparation:

• Lyse cells in a buffer containing protease inhibitors to prevent degradation of ATG4C

• Determine protein concentration and load 20-30 μg of total protein per lane

Electrophoresis and Transfer:

• Separate proteins on 10-12% SDS-PAGE gels (appropriate for the 52 kDa ATG4C protein)

• Transfer to PVDF or nitrocellulose membranes using standard protocols

Antibody Incubation:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary ATG4C antibody at optimal dilution (typically 1:1000, but ranges from 1:200 to 1:20,000 depending on the antibody)

• Incubate overnight at 4°C with gentle agitation

• Wash thoroughly with TBST (3-5 times, 5-10 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody (typically 1:10,000-1:20,000 dilution) for 1 hour at room temperature

• Wash thoroughly with TBST

Detection:

• Develop using ECL substrate or fluorescent-based detection methods

• For dual detection, consider using IRDye-conjugated secondary antibodies (e.g., IRDye 800CW and IRDye 680RD) for multiplex analysis

Controls and Validation:

• Include positive controls (e.g., Jurkat, Raji, or HEK293 cell lysates, which show good expression of ATG4C)

• Include negative controls (e.g., ATG4C knockout cell lysates if available)

• Verify the expected band size of approximately 52-55 kDa

• Note that some antibodies may detect an additional band of unknown origin at 23 kDa

How should researchers optimize immunohistochemistry protocols for ATG4C detection in different tissues?

Optimizing immunohistochemistry protocols for ATG4C detection requires attention to several key factors:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin following standard protocols

• Section tissues at 4-5 μm thickness

Antigen Retrieval (Critical Step):

• Perform heat-mediated antigen retrieval with EDTA buffer (pH 9.0) as this has been shown to be effective for ATG4C detection

• Alternative methods include citrate buffer (pH 6.0), but EDTA buffer may yield superior results for some ATG4C epitopes

Staining Protocol:

• Block endogenous peroxidase activity with 3% hydrogen peroxide

• Apply protein block to reduce non-specific binding

• Incubate with ATG4C primary antibody at optimized dilution (typically 1:20-1:250 for IHC)

• Incubate overnight at 4°C or for 1-2 hours at room temperature

• Wash thoroughly with TBST

• Apply appropriate detection system (polymer-based systems often provide better sensitivity)

• Develop with DAB and counterstain with hematoxylin

Tissue-Specific Considerations:

• Skeletal Muscle: ATG4C is highly expressed in skeletal muscle, making it an excellent positive control tissue

• Liver and Heart: Also show strong ATG4C expression and can serve as positive controls

• Cancer Tissues: May show altered expression patterns, requiring optimization of antibody concentration

Validation Methods:

• Include known positive control tissues (skeletal muscle, heart, liver)

• Perform parallel staining with different ATG4C antibodies targeting different epitopes

• Include peptide competition controls to confirm specificity

• Compare staining patterns with mRNA expression data from publicly available databases

What experimental approaches can researchers use to study the dual function of ATG4C in proteolytic activation and delipidation?

Studying the dual function of ATG4C requires specialized experimental approaches that can distinguish between its proteolytic activation and delipidation activities:

1. In Vitro Enzymatic Assays:

• Proteolytic Activity Assay: Use recombinant ATG8 family proteins (LC3, GABARAP) with C-terminal extensions as substrates and measure cleavage efficiency by ATG4C

• Delipidation Assay: Use PE-conjugated ATG8 proteins and measure the rate of PE removal by ATG4C

• Comparative Analysis: Compare ATG4C activities with ATG4B to highlight functional differences (ATG4C shows weaker proteolytic but stronger delipidation activity)

2. Cellular Assays with Antibody-Based Detection:

• LC3 Processing Analysis: Monitor the conversion of LC3-I to LC3-II and back using Western blotting with LC3 and ATG4C antibodies

• Pulse-Chase Experiments: Track LC3 processing over time with and without ATG4C overexpression or knockdown

• Fluorescent Reporter Systems: Use GFP-LC3 constructs and monitor autophagosome formation and clearance in relation to ATG4C expression

3. Mutational Analysis:

• Create catalytic mutants of ATG4C that selectively affect either proteolytic or delipidation activity

• Use ATG4C antibodies to immunoprecipitate these mutants and assess their interaction with substrate proteins

• Compare the effect of wild-type and mutant ATG4C on autophagy flux

4. Stress-Induced Autophagy Models:

• Since ATG4C function is more prominent under stress conditions , compare its dual activities during:

  • Nutrient starvation

  • Oxidative stress

  • Hypoxia

  • Drug-induced autophagy

5. Advanced Imaging Techniques:

• Co-localization Studies: Use ATG4C antibodies alongside markers for autophagosomes and autolysosomes

• FRET-Based Sensors: Develop sensors that can distinguish between the two enzymatic activities of ATG4C

• Live-Cell Imaging: Monitor ATG4C dynamics during autophagy induction and completion

Data Analysis and Interpretation:

• Quantify both proteolytic and delipidation activities under various conditions

• Compare results with ATG4B to highlight the unique functions of ATG4C

• Correlate enzymatic activities with autophagy flux and cellular outcomes

How can researchers address weak or absent signals when detecting ATG4C?

When encountering weak or absent signals while detecting ATG4C, researchers should consider the following troubleshooting approaches:

Potential Causes and Solutions:

• Low Endogenous Expression:

  • ATG4C expression varies by tissue and cell type; use tissues known to express high levels (skeletal muscle, heart, liver, testis) as positive controls

  • Consider inducing autophagy (starvation, rapamycin treatment) to potentially increase ATG4C expression

  • For cell lines with low expression, consider using concentrated lysates or immunoprecipitation before detection

• Antibody-Related Issues:

  • Verify antibody reactivity to your species of interest

  • Try antibodies targeting different epitopes (N-terminal vs C-terminal)

  • Increase antibody concentration or incubation time

  • Some antibodies may have lot-to-lot variations; validate each new lot

• Protocol Optimization:

  • For Western blotting: Optimize protein loading (20-30 μg typically recommended)

  • For IHC: Test different antigen retrieval methods (EDTA buffer pH 9.0 often works well)

  • For IF: Try different fixation methods (paraformaldehyde vs methanol)

• Sample Preparation Issues:

  • Ensure complete lysis with appropriate buffers containing protease inhibitors

  • Avoid repeated freeze-thaw cycles of samples

  • For tissues, ensure proper fixation and processing

• Detection System Sensitivity:

  • Use more sensitive detection systems (ECL Prime, Super Signal West Femto)

  • For IHC/IF, consider amplification systems (tyramide signal amplification)

  • For challenging samples, try fluorescent-based Western detection methods

What controls should be included when studying ATG4C in different experimental contexts?

Proper controls are essential for reliable ATG4C research across different experimental contexts:

Essential Controls for ATG4C Research:

Context-Specific Controls:

• Cell Type Variation:

  • Include multiple cell lines with known ATG4C expression levels

  • Compare primary cells with immortalized cell lines

• Stress Conditions:

  • Compare basal vs. stressed conditions (ATG4C function is more prominent under stress)

  • Include time course experiments to capture dynamic changes

• Knockout Validation:

  • When using CRISPR/Cas9 or siRNA approaches, verify knockdown/knockout efficiency

  • Use multiple siRNA sequences to control for off-target effects

• Antibody Validation:

  • For new antibodies, include peptide competition assays

  • For established antibodies, compare results with published data

How can researchers differentiate between ATG4C and other ATG4 family members in their experiments?

Differentiating between ATG4C and other ATG4 family members (ATG4A, ATG4B, ATG4D) requires careful experimental design:

Antibody Selection Strategies:

• Epitope Specificity:

  • Choose antibodies raised against unique regions of ATG4C

  • Avoid antibodies targeting conserved catalytic domains if specificity is critical

  • Review sequence alignment of ATG4 family members to identify unique regions

• Validation Methods:

  • Test antibody cross-reactivity against recombinant ATG4A, ATG4B, and ATG4D proteins

  • Use overexpression systems for each family member to confirm specificity

  • Verify specificity in knockout/knockdown systems for each family member

Experimental Approaches:

• mRNA Expression Analysis:

  • Use RT-qPCR with gene-specific primers to quantify each ATG4 family member

  • Compare protein expression (via antibodies) with mRNA levels to confirm identity

• Functional Discrimination:

  • Leverage known functional differences: ATG4C shows weaker proteolytic activity but stronger delipidation activity compared to ATG4B

  • ATG4C is weakly or not involved in phagophore growth during mitophagy, unlike other family members

• Expression Pattern Analysis:

  • ATG4C is highly expressed in skeletal muscle, heart, liver, and testis

  • Different ATG4 family members show distinct tissue expression patterns

• Molecular Weight Differentiation:

  • ATG4A: ~45 kDa

  • ATG4B: ~44 kDa

  • ATG4C: ~52-55 kDa

  • ATG4D: ~39 kDa

• Stress Response Patterns:

  • ATG4C function is more prominent under stress conditions (starvation)

  • Monitor differential responses of ATG4 family members to various autophagy inducers

How might emerging technologies enhance the study of ATG4C using antibody-based approaches?

Emerging technologies promise to advance ATG4C research through improved antibody-based approaches:

Advanced Imaging Technologies:

• Super-Resolution Microscopy:

  • Study ATG4C localization at autophagosome formation sites with nanometer precision

  • Track ATG4C dynamics during autophagy with improved spatial resolution

• Proximity Labeling Techniques:

  • BioID or APEX2 fusions with ATG4C to identify proximal interacting proteins

  • Combine with specific antibodies for validation of interactions

• Live-Cell Imaging Advances:

  • CRISPR-mediated endogenous tagging of ATG4C for physiological expression level imaging

  • Optogenetic control of ATG4C activity combined with antibody-based detection

Antibody Engineering and Single-Cell Technologies:

• Nanobodies and Recombinant Antibody Fragments:

  • Develop smaller ATG4C-specific binding proteins for improved tissue penetration

  • Create intrabodies to track and manipulate ATG4C in living cells

• Single-Cell Proteomics:

  • Analyze ATG4C expression and modification states at single-cell resolution

  • Combine with spatial transcriptomics to correlate ATG4C protein and mRNA levels

• Mass Cytometry (CyTOF):

  • Multiplex ATG4C detection with other autophagy markers

  • Analyze ATG4C in heterogeneous cell populations or tissues

Functional and Structural Approaches:

• CRISPR Screening:

  • Identify genes that modulate ATG4C activity using CRISPR screens combined with antibody-based readouts

  • Create domain-specific ATG4C mutants to dissect structure-function relationships

• Structural Biology Integration:

  • Develop conformation-specific antibodies that recognize active vs. inactive ATG4C

  • Create antibodies against specific post-translational modifications of ATG4C

• Antibody-Drug Conjugates (ADCs):

  • Target autophagy modulation in specific cellular compartments using ATG4C antibodies

  • Develop research tools to manipulate ATG4C activity in specific cell populations

What are the key research questions about ATG4C that remain to be addressed with improved antibody tools?

Despite progress in understanding ATG4C, several key research questions remain that could benefit from improved antibody tools:

Fundamental Biology Questions:

• Regulatory Mechanisms:

  • How is ATG4C activity regulated post-translationally?

  • What protein interactions control ATG4C substrate specificity?

  • Are there tissue-specific cofactors that modulate ATG4C function?

• Substrate Specificity:

  • What determines ATG4C's preference for certain ATG8 family members?

  • Are there non-canonical substrates for ATG4C beyond ATG8 proteins?

  • How does ATG4C distinguish between its roles in processing and delipidation?

• Structural Dynamics:

  • What conformational changes occur during ATG4C activation?

  • How do ATG4C's active sites differ from other ATG4 family members?

  • Can conformation-specific antibodies reveal activation states of ATG4C?

Disease-Related Questions:

• Cancer Biology:

  • How does ATG4C expression correlate with cancer progression across different tumor types?

  • Can ATG4C serve as a prognostic marker or therapeutic target?

  • What is the mechanistic basis for ATG4C's apparent tumor suppressor function?

• Neurodegeneration:

  • Is ATG4C dysregulated in neurodegenerative diseases with autophagy defects?

  • Could ATG4C modulation represent a therapeutic approach for protein aggregation disorders?

• Aging and Longevity:

  • How does ATG4C activity change during aging?

  • Could enhanced ATG4C function promote longevity through improved autophagy?

Methodological Advancements Needed:

• Activity-Based Probes:

  • Development of probes that specifically measure ATG4C enzymatic activity

  • Creation of sensors that distinguish between proteolytic and delipidation functions

• Isoform-Specific Detection:

  • Tools to differentiate between potential splice variants of ATG4C

  • Antibodies that recognize specific post-translational modifications

• In Vivo Imaging:

  • Non-invasive methods to track ATG4C activity in animal models

  • PET ligands based on ATG4C antibodies for translational research