ATG4B Antibody

What is ATG4B and what cellular functions does it perform?

ATG4B, also known as autophagin-1, APG4B, AUTL1, and KIAA0943, belongs to the peptidase C54 family and serves as a cysteine protease essential for autophagy. It has a calculated and observed molecular weight of approximately 44 kDa . ATG4B primarily functions in two critical steps of the autophagy process:

• The proteolytic processing of pro-ATG8 family proteins (including LC3 and GABARAP subfamilies) to expose a C-terminal glycine residue for lipidation

• The delipidation and recycling of ATG8 proteins from autophagosomal membranes

Recent research has shown that ATG4B has significant functions beyond canonical autophagy, including a role in antiviral immunity by regulating TBK1 (TANK binding kinase 1) degradation . ATG4B has been identified as a negative regulator of human antiviral immune responses by targeting TBK1 for autophagic degradation during the advanced stage of viral infection .

What validation methods should I use to confirm ATG4B antibody specificity?

To ensure the validity of your ATG4B antibody results, employ multiple validation techniques:

• Genetic validation: Use ATG4B knockout or knockdown cells as negative controls. The search results indicate various published studies have used ATG4B-deficient cells to validate antibody specificity .

• Molecular weight confirmation: Verify the detection of the expected 44 kDa band in Western blotting applications .

• Cross-reactivity testing: Confirm specificity by testing reactivity with other ATG4 family members (ATG4A, ATG4C, ATG4D). This is particularly important as these proteins share structural similarities.

• Multiple antibody comparison: Use antibodies from different sources or those targeting different epitopes of ATG4B to confirm consistent results.

• Expression pattern analysis: Compare detection with known expression patterns in tissues and cell lines. The antibody has been tested and shown positive Western blot detection in HEK-293, HuH-7, HepG2, Jurkat, and HeLa cells .

What are the optimal protocols for using ATG4B antibodies in Western blotting?

Based on the product information and research applications, the following protocol is recommended for Western blotting:

For optimal results, perform antibody titration to determine the ideal concentration for your specific sample type and detection system. The observed molecular weight should be approximately 44 kDa .

How should I prepare samples for immunohistochemistry with ATG4B antibodies?

The recommended protocol for immunohistochemistry based on published data includes:

• Tissue preparation and fixation: Fix tissues in 10% neutral buffered formalin and embed in paraffin.

• Antigen retrieval options:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

• Endogenous peroxidase blocking: Treat sections with 3% H₂O₂ for 10 minutes at room temperature .

• Antibody dilution and incubation: Dilute ATG4B antibody 1:50-1:500 (with 1:200 being commonly used) and incubate for 12 hours at 4°C .

• Detection system: Use DAB (3,3'-diaminobenzidine) as a chromogenic substrate .

• Scoring system: Quantify results using immunoreactivity scoring with image analysis software such as ImageJ (IHC Profiler) using the formula:
  Score = (Number of pixels in a zone) × (Score of the zone)/Total number of pixels in the image

  The scoring grades are typically divided into 4 levels:

  • High Positive (Score = 4)

  • Positive (Score = 3)

  • Low Positive (Score = 2)

  • Negative (Score = 1)

What are the key considerations for immunoprecipitation with ATG4B antibodies?

Immunoprecipitation studies with ATG4B antibodies require careful attention to several factors:

• Antibody amount: Use 0.5-4.0 μg of antibody for every 1.0-3.0 mg of total protein lysate .

• Cell types: The antibody has been validated for IP in HeLa cells but may work with other cell types .

• Lysis conditions: Use a mild lysis buffer (such as NP-40 or CHAPS-based) to preserve protein-protein interactions if studying ATG4B binding partners.

• Pre-clearing step: Include a pre-clearing step with protein A/G beads to reduce non-specific binding.

• Controls: Include an IgG control from the same species as the ATG4B antibody.

• Elution: Perform elution under non-denaturing conditions if downstream functional assays are planned.

• Co-IP applications: The antibody has been cited for Co-IP applications in previous studies, making it suitable for investigating ATG4B interactions with other proteins .

How can ATG4B antibodies be used to investigate its role in antiviral immunity?

Recent research has revealed ATG4B's role as a negative regulator of antiviral immunity through GABARAP-directed degradation of TBK1 . To investigate this function, researchers can:

• Monitor ATG4B-TBK1 interaction: Use co-immunoprecipitation with the ATG4B antibody to pull down TBK1 and analyze their physical interaction under different viral infection conditions.

• Analyze TBK1 degradation: Compare TBK1 protein levels in control versus ATG4B-deficient cells during viral infection using Western blotting. Research shows that ATG4B-deficient cells presented increased protein abundance of TBK1 both in uninfected and virus-infected conditions .

• Evaluate antiviral response: Measure type I interferon production in the presence or absence of ATG4B during viral infection. Flow cytometry analyses have shown that the percentage of virus-infected cells (GFP+) was considerably decreased in ATG4B-deficient cells compared to control cells .

• Assess ATG4B inhibitor effects: Test the impact of ATG4B inhibitors like S130 on viral replication and immune response. S130 has been shown to block ATG4B-dependent autophagic degradation of TBK1 and contribute to host defense against viral infection .

• Examine GABARAP involvement: Investigate the role of GABARAP in this process, as ATG4B serves as an adaptor bridging TBK1 to GABARAP, leading to TBK1-GABARAP interaction for autophagic degradation .

What is the relationship between ATG4B and cancer, and how can antibodies help study this connection?

High ATG4B expression has been correlated with poor survival of patients with gastric cancer and has been found essential for tumor growth . Researchers can use ATG4B antibodies to:

• Expression profiling: Perform immunohistochemistry on tumor samples to assess ATG4B expression levels and correlate with clinical outcomes. The recommended antibody dilution for IHC is 1:50-1:500 .

• Prognostic marker evaluation: Analyze ATG4B expression in patient samples to evaluate its potential as a prognostic biomarker for cancer progression.

• Therapeutic target assessment: Measure changes in ATG4B expression or activity in response to anti-cancer treatments or ATG4B inhibitors.

• Functional studies: Use the antibody for knockdown validation in functional studies examining the role of ATG4B in cancer cell growth, migration, and invasion.

• Autophagy dependency: Investigate whether cancer cells are dependent on ATG4B-mediated autophagy for survival under stress conditions.

How can I distinguish between ATG4B and other ATG4 family members using antibodies?

The mammalian ATG4 family consists of four members (ATG4A, ATG4B, ATG4C, and ATG4D) with distinct but overlapping functions. To distinguish between them:

• Epitope selection: Use antibodies targeting unique regions that don't share homology with other ATG4 family members.

• Validation in knockout models: Test the antibody in cells deficient for specific ATG4 family members. For example, the search results discuss ATG4A-only mice that are deficient in ATG4B, ATG4C, and ATG4D .

• Expression pattern analysis: Different ATG4 family members may have distinct tissue expression patterns. For example, ATG4D has been associated with cerebellar neurodegeneration in knockout studies .

• Functional assay correlation: ATG4B shows preference for certain ATG8 family members. ATG4A appears less effective at cleaving LC3A, LC3B, GABARAP, and GABARAPL1, but more effective with GABARAPL2 .

• Molecular weight distinction: Although there are slight differences in molecular weight between ATG4 family members, high-resolution gels may be needed to distinguish them effectively.

What are common challenges when using ATG4B antibodies and how can they be addressed?

Researchers may encounter several issues when using ATG4B antibodies:

• Non-specific bands in Western blot:

  • Solution: Optimize antibody dilution (1:500-1:3000 recommended)

  • Use longer blocking times and more stringent washing protocols

  • Include appropriate positive controls (HEK-293, HuH-7, HepG2, Jurkat, or HeLa cells)

• Weak or no signal in immunohistochemistry:

  • Solution: Test different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Adjust antibody concentration (1:50-1:500)

  • Extend incubation time to 12 hours at 4°C

• Insufficient immunoprecipitation yield:

  • Solution: Adjust antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Optimize lysis buffer composition

  • Extend incubation time with antibody

• Cross-reactivity with other ATG4 family members:

  • Solution: Use genetic knockouts as controls

  • Perform parallel experiments with antibodies specific to other ATG4 family members

• Variable results across different samples:

  • Solution: Normalize loading with proper housekeeping controls

  • Titrate antibody concentration for each specific sample type or tissue

  • Follow the recommendation that "this reagent should be titrated in each testing system to obtain optimal results"

How should I design experiments to study ATG4B activity and function?

When designing experiments to study ATG4B:

• Functional assays: ATG4B functions as a cysteine protease that cleaves pro-LC3/GABARAP proteins and deconjugates lipidated LC3/GABARAP from autophagosomal membranes. Design experiments to monitor:

  • Processing of pro-LC3/GABARAP to their mature forms

  • Delipidation of LC3-II/GABARAP-II

• Genetic manipulation:

  • Use ATG4B knockout or knockdown approaches

  • Consider the functional redundancy with other ATG4 family members

  • Study mice deficient in ATG4B (ATG4B-/-) which show limited autophagic competency but near-normal lifespan when unchallenged

• Drug intervention:

  • Apply ATG4B inhibitors like S130 to block its function

  • Monitor effects on autophagy, antiviral response, or tumor growth

• Stress conditions:

  • ATG4B-/- mice are unable to mount appropriate autophagic responses upon autophagy-inducing stimuli

  • Include starvation, oxidative stress, or other autophagy inducers in experimental design

• Interaction studies:

  • Investigate ATG4B interactions with TBK1 and GABARAP in antiviral immunity

  • Study how ATG4B regulates selective autophagy substrates (p62/SQSTM1, OPTN, NDP52, NBR1)

What is known about ATG4B inhibitors and their research applications?

The small molecule S130 has been identified as an ATG4B inhibitor with significant research implications:

• Mechanism of action: S130 strongly suppresses the cleavage activity of ATG4B, blocking ATG4B-dependent autophagic degradation of TBK1 .

• Antiviral effects: S130 contributes to host defense against viral infection, increasing antiviral response and attenuating VSV infection both in vitro and in vivo .

• Therapeutic potential: S130 has been proposed as a potential therapeutic reagent against virus infection and insufficient immune responses .

• Research applications:

  • Use as a tool compound to study ATG4B-dependent processes

  • Investigate the impact of pharmacological ATG4B inhibition compared to genetic knockout

  • Explore differential effects on various ATG8 family proteins

• Future directions: Development of more specific and potent ATG4B inhibitors may provide valuable tools for both research and potential therapeutic applications.

How does ATG4B function differ among various tissues and pathological conditions?

ATG4B exhibits context-dependent functions across different tissues and disease states:

• Tissue-specific roles:

  • ATG4B antibodies have detected expression in various tissues, including pancreas cancer tissue and mouse heart tissue through IHC

  • ATG4B-/- mice show increased susceptibility to diverse experimentally-induced pathologies in specific tissues, including:

    • Bleomycin-induced pulmonary fibrosis

    • Lung injury during endotoxemia

    • Susceptibility to experimental colitis

    • Cellular damage upon optic nerve transection

    • Obesity and other metabolic disturbances

• Cancer context:

  • High ATG4B expression correlates with poor survival in gastric cancer patients

  • ATG4B appears essential for tumor growth in certain cancer types

  • Research suggests differential roles in various cancer types

• Immune response:

  • ATG4B negatively regulates type I IFN signaling and antiviral immunity

  • Functions as an adaptor for recruiting TBK1 to GABARAP

  • May have distinct functions in different immune cell types

• Aging and neurodegeneration:

  • Triple knockout mice lacking ATG4B/C/D (ATG4A-only) develop multiple aging-related features

  • These mice show accumulation of DNA-damage, inflammation, and senescence markers

  • They exhibit dramatically reduced lifespan compared to wild-type or single knockout animals