ATG5 Antibody

What is ATG5 and what are its primary functions in cellular processes?

ATG5 (also known as APG5L) plays crucial roles in multiple cellular processes. Its primary function involves autophagic vesicle formation, where it conjugates with ATG12 through a ubiquitin-like conjugating system involving ATG7 as an E1-like activating enzyme and ATG10 as an E2-like conjugating enzyme. This ATG12-ATG5 conjugate functions as an E3-like enzyme required for lipidation of ATG8 family proteins and their association with vesicle membranes .

Beyond autophagy, ATG5 is involved in:

• Mitochondrial quality control after oxidative damage

• Lymphocyte development and survival (both B and T cells)

• Antigen processing and presentation for MHC II

• Maintenance of axon morphology and membrane structures

• Adipocyte differentiation

• Primary ciliogenesis through removal of OFD1 and degradation of IFT20

ATG5 also plays a role in apoptotic processes, with its expression occurring relatively late, downstream of caspase activity .

What applications can ATG5 antibodies be used for in research protocols?

ATG5 antibodies can be utilized in multiple research applications with specific recommended protocols:

For optimal immunohistochemistry results, antigen retrieval with TE buffer pH 9.0 is recommended, although citrate buffer pH 6.0 may be used alternatively .

ATG5 antibodies have been validated in multiple cell lines including HeLa, Jurkat, HepG2, A549, NIH/3T3, RAW 264.7, HSC-T6, and PC-12 cells for Western blot, and in human ovary tumor tissue for immunohistochemistry .

What is the molecular weight of ATG5 and how does this affect antibody detection?

ATG5 presents an interesting case for antibody detection:

The discrepancy between calculated and observed molecular weights is likely due to post-translational modifications or detection of the ATG12-ATG5 conjugate . Researchers should be aware that truncated forms of ATG5, such as the 24-kDa calpain-cleaved variant, may exhibit different functions from the full-length protein and may not interact with the same binding partners .

When selecting antibodies, researchers should verify which form(s) of ATG5 are recognized by the specific antibody.

How should ATG5 antibodies be stored for optimal performance?

To maintain optimal antibody performance, follow these storage guidelines:

• Store at -20°C

• Stable for one year after shipment

• Aliquoting is unnecessary for -20°C storage

• Storage buffer typically contains PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Some preparations (20μl sizes) may contain 0.1% BSA

Proper storage ensures antibody stability and consistent performance in experimental applications.

What are the recommended positive controls for ATG5 antibody validation?

Based on validated research protocols, the following positive controls are recommended:

For comprehensive validation, include both cell lines known to express ATG5 and those with ATG5 knockdown or knockout as negative controls .

How does ATG5 contribute to antigen presentation in dendritic cells?

ATG5 plays a critical role in antigen presentation by dendritic cells (DCs), particularly for processing extracellular antigens:

Research using conditional knockout mice (CD11c-Cre × ATG5^flox/flox) has demonstrated that ATG5 is essential for optimal processing and presentation of phagocytosed antigens containing TLR agonists . While innate immune recognition, antigen capture, migration, maturation, and cytokine production remained intact in ATG5-deficient DCs, these cells showed impaired ability to process and present various forms of phagocytosed antigens to CD4+ T cells .

The functional significance of this impairment is substantial:

• In vivo studies showed reduced IFN-γ secretion from CD4+ T cells after HSV-2 infection

• Mice lacking ATG5 in DCs showed more severe disease and succumbed to HSV-2 infection more rapidly

• The defect is specific to processing extracellular antigens, as direct peptide presentation remains intact

This evidence establishes ATG5 as a critical component for antigen processing pathways in DCs, particularly for stimulating protective CD4+ T cell responses against pathogens.

What is the role of nuclear ATG5 in mitotic catastrophe following DNA damage?

Beyond its cytoplasmic role in autophagy, ATG5 has a distinct nuclear function related to mitotic catastrophe:

Following DNA damage by agents like etoposide, ATG5 is upregulated and accumulates in the nucleus through a mechanism involving overlapping nuclear export signal (NES) and nuclear localization signal (NLS) sequences . This nuclear accumulation of ATG5 is both necessary and sufficient to induce mitotic catastrophe in an autophagy-independent manner .

The mechanism involves:

• Physical interaction between ATG5 and survivin in the nucleus

• Disruption of the chromosomal passenger complex by reducing survivin-Aurora B interaction

• Mislocalization of both survivin and Aurora B during mitosis

• Severe disturbances in chromosome alignment and segregation

This dual role of ATG5 represents an important consideration for cancer research, especially when studying responses to DNA-damaging chemotherapeutic agents.

How can researchers distinguish between autophagy-dependent and autophagy-independent functions of ATG5?

Distinguishing between these functions requires strategic experimental approaches:

Research shows that ATG5-K130R can interact with survivin and induce mitotic catastrophe, while ATG5-ΔNES cannot interact with survivin, suggesting the interaction occurs in the nucleus or depends on the leucine-rich sequence modified in this mutant .

What experimental controls should be included when studying ATG5 in antigen presentation?

When investigating ATG5's role in antigen presentation, include these essential controls:

• Cell-type specificity controls:

  • Compare conventional DCs (cDCs) with other antigen-presenting cells

  • Include ATG5-sufficient and ATG5-deficient cells from the same lineage

• Functional pathway controls:

  • Examine direct peptide presentation (should be intact in ATG5-deficient cells)

  • Test different forms of antigen (soluble vs. particulate)

  • Include TLR agonist-containing and TLR agonist-free antigens

• In vivo relevance controls:

  • Measure downstream T cell activation markers (proliferation, cytokine production)

  • Assess protective immunity in infection models (e.g., HSV-2 challenge model)

  • Track disease progression parameters in pathogen challenge models

These controls help establish the specificity of ATG5's contribution to antigen presentation pathways and distinguish it from general defects in DC function.

How does ATG5 interact with survivin and affect the chromosomal passenger complex?

The interaction between ATG5 and survivin represents a critical mechanism for ATG5's role in mitotic catastrophe:

Coimmunoprecipitation studies demonstrate that ATG5 physically interacts with survivin following DNA damage or ectopic ATG5 overexpression . This interaction occurs specifically in the nucleus, as:

• ATG5-ΔNES (which cannot enter the nucleus) fails to interact with survivin

• ATG5-K130R (autophagy-deficient but nucleus-competent) maintains the interaction

• Calpain-cleaved 24-kDa truncated ATG5 cannot interact with survivin

The functional consequence of this interaction is a reduced association between survivin and Aurora B, disrupting the chromosomal passenger complex. Importantly:

• ATG5 is not detectable in Aurora B immunoprecipitates

• The amount of borealin (another chromosomal passenger complex protein) remains unaffected

• These changes lead to mislocalization of Aurora B and survivin during mitosis

• The result is severe disturbance in chromosome alignment and segregation

This mechanism explains how nuclear ATG5 induces mitotic catastrophe independent of its autophagy function.

What is the significance of ATG5 in cancer treatment response?

ATG5's dual role in autophagy and mitotic catastrophe has important implications for cancer therapy:

Research shows that ATG5 is upregulated in cancer cells following treatment with DNA-damaging drugs, both under in vitro and in vivo conditions . This upregulation appears to promote mitotic catastrophe through nuclear accumulation of ATG5 and its interaction with survivin .

The balance between these functions affects therapeutic outcomes:

• Autophagy may initially protect cells from death despite mitotic catastrophe

• Pharmacological inhibition of autophagy in cells with nuclear ATG5 accumulation leads to rapid caspase-dependent cell death

This suggests potential therapeutic strategies:

• Enhancing nuclear ATG5 accumulation to promote mitotic catastrophe

• Combining DNA-damaging agents with autophagy inhibitors to convert mitotic catastrophe to cell death

• Developing approaches to specifically disrupt ATG5-survivin interactions in cancer cells

Understanding this dual role of ATG5 could help explain variable responses to DNA-damaging chemotherapeutics and inform more effective combination treatment strategies.

What methodologies can be used to study ATG5 translocation between cytosol and nucleus?

Several complementary approaches can effectively monitor ATG5's subcellular localization:

Research has shown that upon etoposide treatment or ectopic ATG5 overexpression, significant levels of ATG5 appear in the nucleus where it colocalizes with survivin. In contrast, in cells expressing ATG5-ΔNES, the protein remains exclusively cytosolic with little evidence of survivin colocalization .

How do different post-translational modifications affect ATG5 function?

ATG5 undergoes several post-translational modifications that influence its function:

The balance of these modifications determines whether ATG5 functions in autophagy (primarily the ATG12-conjugated form in the cytoplasm) or in nuclear processes like mitotic catastrophe (free ATG5 in the nucleus) .

Understanding these modifications provides opportunities for selectively targeting specific ATG5 functions in experimental or therapeutic contexts.