ATG13B Antibody

What is ATG13 and what role does its phosphorylation at S318 play in autophagy?

ATG13 (Autophagy-related protein 13) is a critical component of the ULK1 complex that initiates autophagosome formation. The phosphorylation of ATG13 at serine 318 (S318) is particularly important as it represents a regulatory mechanism in the autophagy pathway. This post-translational modification occurs in response to upstream signals and affects ATG13's ability to form complexes with other autophagy-related proteins. Phosphorylation at S318 is considered a marker for activation status within autophagy signaling networks. When investigating autophagy regulation, monitoring this specific phosphorylation site provides more precise information than tracking total ATG13 levels alone, making phospho-specific antibodies valuable research tools for studying the dynamic regulation of autophagy .

How does ATG13 relate to the broader autophagy machinery and antiviral responses?

ATG13 functions within a complex network of autophagy-related proteins that orchestrate autophagosome formation. Recent research shows interconnections between autophagy machinery and antiviral immunity. While ATG13 itself has not been directly implicated in antiviral responses based on the provided search results, the related autophagy protein ATG4B has been identified as a negative regulator of antiviral immunity. ATG4B targets TBK1 (TANK binding kinase 1)—an essential kinase in antiviral signaling—for autophagic degradation during advanced stages of viral infection. This mechanism represents an important regulatory checkpoint preventing excessive immune responses . Understanding these interconnections helps researchers interpret ATG13 phosphorylation status in the context of both canonical autophagy and potential immune response modulation.

What are the key technical specifications of commercially available ATG13 phospho S318 antibodies?

Commercial ATG13 phospho S318 antibodies are typically rabbit polyclonal antibodies generated through repeated immunizations with synthetic peptides corresponding to regions near S318 of human ATG13. These antibodies undergo rigorous purification processes, including affinity purification from monospecific antiserum and cross-adsorption against the non-phosphorylated form of the immunizing peptide. This processing ensures high specificity for the phosphorylated form of ATG13. According to product specifications, reactivity with non-phosphorylated human ATG13 is minimal as evaluated by ELISA and western blot techniques. BLAST analysis typically indicates cross-reactivity with human ATG13 based on 100% sequence homology with the immunogen, though reactivity against homologues from other species may vary by manufacturer .

What are the optimal conditions for using ATG13 phospho S318 antibody in western blot applications?

For western blot applications using ATG13 phospho S318 antibody, researchers should expect to detect a band at approximately 56.6 kDa corresponding to human phosphorylated ATG13 protein. Optimal western blot conditions typically include:

When optimizing western blot conditions, it's crucial to preserve phosphorylation status throughout the experimental procedure. Use fresh phosphatase inhibitors in all buffers and avoid multiple freeze-thaw cycles of samples. Consider using phospho-protein specific staining methods or parallel blots with total ATG13 antibody to confirm specificity and normalize phosphorylation levels .

How can researchers effectively use ATG13 phospho S318 antibody in immunofluorescence microscopy?

For immunofluorescence (IF) applications, ATG13 phospho S318 antibody can be effectively used to visualize the subcellular localization of phosphorylated ATG13 during autophagy induction. The following methodological considerations are important:

• Fixation method: 4% paraformaldehyde is preferred (10-15 minutes at room temperature) as it better preserves phospho-epitopes compared to methanol fixation.

• Permeabilization: Use 0.1-0.2% Triton X-100 for 5-10 minutes; avoid harsher detergents that may extract phosphorylated proteins.

• Blocking: 5% normal serum (from the species of secondary antibody) with 1% BSA in PBS is recommended to minimize background.

• Antibody dilution: Start with 1:100-1:500 dilution and optimize based on signal-to-noise ratio.

• Co-staining markers: Consider co-staining with autophagosome markers like LC3 or other ULK1 complex components to confirm biological relevance.

• Controls: Include cells treated with phosphatase to demonstrate specificity for the phosphorylated form.

• Image acquisition: Use confocal microscopy to precisely visualize punctate structures associated with early autophagosome formation sites.

When interpreting IF results, phosphorylated ATG13 typically shows a punctate pattern upon autophagy induction, reflecting its recruitment to pre-autophagosomal structures .

How should researchers design experiments to study the dynamic phosphorylation of ATG13 at S318?

When designing experiments to study dynamic phosphorylation of ATG13 at S318, researchers should implement time-course analyses with appropriate controls:

• Induction conditions: Include classical autophagy inducers (rapamycin, starvation, Torin1) alongside relevant physiological stimuli for your research question.

• Time points: Capture both early phosphorylation events (5-15 minutes) and later time points (30 minutes to 2 hours) to observe the full phosphorylation dynamic.

• Inhibitor controls: Include mTOR pathway inhibitors (rapamycin) and activators (insulin) to confirm pathway specificity.

• Genetic controls: When possible, include ATG13 S318A mutants (which cannot be phosphorylated at this site) to validate antibody specificity.

• Quantification: Apply densitometric analysis for western blots, normalizing phospho-S318 signal to total ATG13 levels.

• Complementary approaches: Combine antibody-based detection with mass spectrometry-based phosphoproteomics for validation of specific modifications.

• Kinase inhibitor panel: Consider using a panel of kinase inhibitors to identify the specific kinase responsible for S318 phosphorylation in your experimental system.

The experimental design should also account for cell type-specific variations in ATG13 phosphorylation kinetics and consider the effects of cell confluence and passage number on basal autophagy levels .

What considerations are important when using ATG13 phospho S318 antibody in flow cytometry applications?

Flow cytometry applications with ATG13 phospho S318 antibody require specific methodological considerations since this targets an intracellular phospho-epitope:

• Fixation and permeabilization: Use formaldehyde fixation (2-4%) followed by methanol permeabilization to access intracellular antigens while preserving phospho-epitopes.

• Buffer composition: Include phosphatase inhibitors in all buffers to maintain phosphorylation status.

• Antibody validation: Perform parallel western blot analysis to confirm specificity before flow cytometry applications.

• Controls:

  • Isotype controls to establish background fluorescence

  • Phosphatase-treated samples as negative controls

  • Samples with known ATG13 phosphorylation status as positive controls

• Co-staining considerations: When performing multi-parameter analysis, optimize fluorophore combinations to avoid spectral overlap with common autophagy markers.

• Gating strategy: Develop a consistent gating strategy that accounts for cell cycle-dependent variations in autophagy.

• Data interpretation: Quantify shifts in median fluorescence intensity rather than percentage of positive cells when analyzing phosphorylation events.

Flow cytometry offers the advantage of analyzing ATG13 phosphorylation at the single-cell level, revealing potential heterogeneity in autophagy activation across cell populations that might be masked in bulk western blot analyses .

How can researchers troubleshoot weak or absent signals when using ATG13 phospho S318 antibody in western blot?

When encountering weak or absent signals with ATG13 phospho S318 antibody in western blot applications, consider the following troubleshooting approaches:

It's particularly important to remember that phosphorylation events can be transient and sensitive to experimental conditions. Optimal stimulation conditions might vary between cell types, and the timing of phosphorylation events should be carefully determined through preliminary time-course experiments. Additionally, ensure that your positive control conditions effectively induce autophagy in your specific cell system, as autophagy induction pathways can vary between cell types .

How should researchers interpret contradictory data between ATG13 phosphorylation and other autophagy markers?

When facing contradictory data between ATG13 phosphorylation and other autophagy markers, consider these analytical approaches:

• Pathway crosstalk analysis: ATG13 phosphorylation may be affected by multiple signaling pathways beyond canonical autophagy. Examine potential crosstalk with mTOR, AMPK, and ULK1 pathways.

• Temporal considerations: Different autophagy markers represent distinct stages of the process. ATG13 phosphorylation is an early event, while LC3-II formation or p62 degradation occurs later. Misaligned time points may explain apparent contradictions.

• Context-dependent regulation: Consider cell type-specific regulatory mechanisms. Different tissues may exhibit unique ATG13 phosphorylation patterns relative to other autophagy markers.

• Autophagy-independent functions: Emerging research suggests ATG proteins may have functions outside canonical autophagy. ATG13 phosphorylation might reflect these alternative roles in certain contexts.

• Technical validation: Confirm findings using orthogonal methods (e.g., IF, flow cytometry) and alternative antibodies targeting different ATG13 epitopes.

• Flux considerations: Autophagy is a dynamic process, and static measurements can be misleading. Consider using flux assays with lysosomal inhibitors to distinguish between increased autophagy initiation and impaired autophagosome clearance.

• Genetic validation: Use genetic approaches (siRNA knockdown, CRISPR knockout, or phospho-mutants) to validate the functional significance of observed phosphorylation events.

Remember that autophagy regulation is highly complex and context-dependent, and seemingly contradictory data may reveal novel regulatory mechanisms rather than experimental errors .

How does ATG13 phosphorylation relate to recent discoveries regarding autophagy in antiviral immunity?

Recent research has expanded our understanding of the interconnections between autophagy machinery and antiviral immunity. While the search results don't directly link ATG13 phosphorylation to antiviral responses, they reveal important connections between autophagy components and immune function that researchers should consider when studying ATG13:

• Regulatory mechanisms: The autophagy-related cysteine protease ATG4B has been identified as a negative regulator of antiviral immunity by targeting TBK1 (TANK binding kinase 1) for autophagic degradation during viral infection. This mechanism represents a potential feedback loop that prevents excessive immune activation .

• Adaptor functions: ATG4B serves as an adaptor for recruiting TBK1 to GABARAP (GABA type A receptor-associated protein), facilitating TBK1-GABARAP interaction through the LC3-interacting region (LIR) motif of TBK1's ULD domain .

• Pharmacological implications: Small-molecule inhibitors of autophagy components, such as the ATG4B inhibitor S130, can enhance antiviral responses by blocking the autophagic degradation of immune signaling components like TBK1 .

• Viral countermeasures: Some viruses exploit autophagy machinery for their replication. For example, ATG4B has been shown to hydrolyze the polyprotein of enterovirus 71 (EV71), aiding viral replication .

These findings suggest that researchers studying ATG13 phosphorylation should consider potential roles beyond canonical autophagy, particularly in contexts involving viral infection or immune activation. Future studies might explore whether ATG13 phosphorylation status affects its potential interactions with immune signaling components or viral proteins .

What are the latest methodological approaches for studying ATG13 phosphorylation in complex experimental systems?

Advanced methodological approaches for studying ATG13 phosphorylation in complex experimental systems include:

• Proximity labeling techniques: BioID or APEX2 fusion proteins can identify transient interaction partners of phosphorylated ATG13 in living cells, revealing context-specific signaling networks.

• Phospho-proteomic mass spectrometry: Targeted mass spectrometry approaches enable precise quantification of multiple phosphorylation sites on ATG13 simultaneously, revealing potential crosstalk between different regulatory events.

• Genetic code expansion: Site-specific incorporation of phosphomimetic amino acids through amber codon suppression allows creation of constitutively "phosphorylated" ATG13 to study functional consequences.

• Optogenetic tools: Light-inducible kinase systems permit spatiotemporal control of ATG13 phosphorylation, enabling studies of localized autophagy activation.

• CRISPR-based screening: Genome-wide or targeted CRISPR screens can identify novel regulators of ATG13 phosphorylation under different physiological conditions.

• Bispecific antibody applications: As described in search result , bispecific antibodies that simultaneously recognize phosphorylated ATG13 and another autophagy component could enable more precise tracking of autophagy complexes.

• Live-cell imaging approaches: FRET-based biosensors can provide real-time visualization of ATG13 phosphorylation dynamics in living cells.

• In situ proximity ligation assay (PLA): This technique allows visualization of the interaction between phosphorylated ATG13 and its binding partners directly in fixed cells, providing spatial information about signaling events.

These advanced methodologies enable researchers to move beyond static measurements of ATG13 phosphorylation toward understanding its dynamic regulation in complex biological contexts .