STX17 Antibody

What is STX17 and why is it important in cellular research?

STX17 (Syntaxin 17) is a SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein critically involved in membrane fusion processes. In humans, the canonical protein has 302 amino acid residues with a molecular weight of approximately 33.4 kDa . It plays essential roles in autophagy, particularly in autophagosome-lysosome fusion, and has been implicated in early secretory pathway functions including maintenance of endoplasmic reticulum-Golgi intermediate compartment architecture . STX17's importance stems from its dual function in both autophagy initiation and autophagosome-lysosome fusion, making it a key regulatory node in cellular degradation pathways central to numerous physiological and pathological processes .

What are the primary applications of STX17 antibodies in research?

STX17 antibodies are primarily utilized for:

• Western Blotting (WB): Most commonly used to detect STX17 protein expression and quantify levels in different experimental conditions

• Immunohistochemistry (IHC): Visualizing STX17 distribution in tissue samples

• Immunocytochemistry (ICC) and Immunofluorescence (IF): Examining subcellular localization and dynamics

• Immunoprecipitation (IP): Isolating STX17 and its interacting partners

Over 130 citations in scientific literature describe the use of STX17 antibodies, with Western Blot being the most widely employed application . These antibodies enable researchers to investigate STX17's roles in autophagy, membrane trafficking, and related cellular processes across multiple experimental systems.

What species reactivity should be considered when selecting STX17 antibodies?

When selecting STX17 antibodies, researchers should consider cross-reactivity with relevant experimental models. STX17 is highly conserved across species, with orthologs identified in mouse, rat, bovine, zebrafish, chimpanzee, and chicken . Most commercial antibodies are validated for human, mouse, and rat reactivity . For studies involving other species, it's advisable to perform preliminary validation experiments. Sequence alignment analysis can help predict cross-reactivity based on epitope conservation. Importantly, when studying STX17 in evolutionary contexts, consider that while the core SNARE domain is highly conserved, the regulatory regions may show greater variation across species, potentially affecting antibody recognition .

How should STX17 antibodies be validated before use in autophagy research?

For rigorous validation of STX17 antibodies in autophagy research, implement this multi-step approach:

• Knockout/knockdown controls: Essential validation includes testing the antibody in STX17 knockout or knockdown cells to confirm specificity .

• Phospho-specific validation: For phospho-specific antibodies (e.g., pS202), pretreat samples with λ phosphatase as a negative control .

• Expression system testing: Overexpression of tagged STX17 constructs can serve as positive controls.

• Cross-reactivity assessment: Particularly important when studying multiple SNARE proteins simultaneously.

• Application-specific validation: Different applications (WB, IF, IHC) may require distinct validation strategies.

An example validation protocol involves parallel immunoblotting of wild-type and STX17-depleted samples, followed by immunofluorescence comparison to confirm absence of signal in knockout models across all intended applications .

What are the optimal fixation and permeabilization conditions for STX17 immunostaining?

Based on published protocols, optimal conditions for STX17 immunostaining include:

• Fixation: 4% paraformaldehyde in phosphate buffer for 10 minutes at room temperature preserves STX17 epitopes while maintaining cellular architecture .

• Permeabilization: Mild permeabilization with 50 μg/ml digitonin in PBS for 5 minutes is recommended over stronger detergents like Triton X-100, as it better preserves membrane structures where STX17 resides .

• Blocking: 3% BSA in PBS for 10 minutes before primary antibody incubation reduces background signal .

• Antibody incubation: Extended primary antibody incubation (16 hours at 4°C) followed by secondary antibody incubation for 1 hour at room temperature optimizes signal-to-noise ratio .

These conditions are particularly important when studying membrane-associated proteins like STX17, as overly harsh permeabilization can disrupt membrane structures and create artifacts in localization studies.

How can I differentiate between different subcellular pools of STX17?

Differentiating between subcellular pools of STX17 requires careful methodological considerations:

• Co-localization studies: Use established markers for:

  • ER: Calnexin or PDI

  • Mitochondria: TOM20 or MitoTracker

  • Golgi: GM130

  • Autophagosomes: LC3

  • ER-mitochondria contact sites: Specialized markers

• Phospho-specific antibodies: Phosphorylated STX17 (pS202) localizes specifically to the Golgi apparatus and can be detected with phospho-specific antibodies .

• Subcellular fractionation: Complement imaging with biochemical isolation of organelles to quantify STX17 distribution.

• Live-cell imaging: Tagged STX17 constructs can be used for real-time tracking of dynamic redistribution during autophagy induction.

• Super-resolution microscopy: Techniques like STORM or STED provide enhanced resolution for distinguishing closely positioned membrane structures.

It's important to note that STX17 has been reported in various membranes including ER, mitochondria, ER-mitochondria contact sites, and its distribution changes dynamically during autophagy induction .

How does STX17 phosphorylation status affect its function and detection?

STX17 phosphorylation critically regulates its function and subcellular localization, with important implications for detection strategies:

• Functional impact: TBK1-mediated phosphorylation of STX17 at S202 controls autophagy initiation by regulating the formation of ATG13-containing protein complexes . This represents a distinct regulatory mechanism from STX17's role in autophagosome-lysosome fusion.

• Localization effects: Phosphorylated STX17 (pS202) specifically localizes to the Golgi apparatus, whereas non-phosphorylated STX17 exhibits broader distribution across various membrane compartments .

• Detection considerations:

  • Phospho-specific antibodies (e.g., against pS202) enable specific detection of the active Golgi-associated pool

  • Phosphatase treatment controls are essential for validating phospho-specific antibody signals

  • TBK1 knockout cells show dramatically reduced pS202 signal, though some residual phosphorylation persists

• Experimental design implications: Studies focusing on STX17's role in autophagy should account for these distinct phosphorylation-dependent functions, potentially using phosphomimetic or phospho-deficient mutants to dissect specific functions.

What are the key factors influencing STX17 recruitment to mature autophagosomes?

Recent research has uncovered several critical factors that regulate the temporal recruitment of STX17 to mature autophagosomes:

• Electrostatic interactions: STX17 recruitment depends on positively charged amino acids in its C-terminal region that interact with increasingly negatively charged autophagosomal membranes during maturation .

• Phospholipid composition: Phosphatidylinositol 4-phosphate (PI4P) accumulates during autophagosome maturation and is required for STX17 recruitment . In vitro experiments demonstrate that PI4P dephosphorylation by recombinant Sac1PD significantly impairs STX17 binding to isolated autophagosomes.

• Phosphorylation regulation: While distinct from membrane recruitment mechanisms, TBK1-mediated phosphorylation of STX17 at S202 affects its function in autophagy initiation .

• Potential co-factors: Some studies suggest roles for LC3/GABARAP family proteins and immunity-related GTPase M (IRGM) in STX17 recruitment, although findings are inconsistent across studies .

• Membrane curvature: The hairpin-like C-terminal transmembrane domains of STX17 may sense membrane curvature changes during autophagosome maturation.

This temporally regulated recruitment ensures STX17 participates in fusion only after complete autophagosome closure, preventing premature fusion that could release lysosomal enzymes into the cytosol .

What experimental approaches can determine STX17-interacting partners during autophagy?

To comprehensively identify and characterize STX17-interacting partners during autophagy, researchers can employ multiple complementary approaches:

• Proximity labeling techniques:

  • BioID or TurboID fused to STX17 can identify proximal proteins in living cells

  • APEX2-based proximity labeling offers temporal resolution for capturing dynamic interactions

• Co-immunoprecipitation strategies:

  • Standard co-IP using STX17 antibodies followed by mass spectrometry

  • Crosslinking-assisted immunoprecipitation to capture transient interactions

  • Comparison between basal, autophagy-induced, and autophagy-blocked conditions

• SNARE complex analysis:

  • Non-denaturing gel electrophoresis to preserve SNARE complexes

  • Sequential co-IP focusing on known SNARE partners (SNAP29, VAMP7/8)

  • In vitro reconstitution assays with purified components

• Microscopy-based interaction studies:

  • FRET or BRET analysis for direct protein interactions

  • Split-fluorescent protein complementation assays

  • Co-localization with super-resolution microscopy

• Perturbation approaches:

  • Mutagenesis of key domains (particularly the SNARE domain and regulatory regions)

  • Comparison of wild-type vs. phospho-mutant interactomes

These approaches have revealed key STX17 interactions with SNAP29 and VAMP7/8 in autophagosome-lysosome fusion, while also identifying roles for YKT6 as a complementary autophagosomal SNARE protein .

Why might STX17 antibodies show inconsistent results in different experimental conditions?

Several factors can contribute to inconsistent STX17 antibody performance across experiments:

• Epitope accessibility issues:

  • STX17's conformation changes during SNARE complex formation, potentially masking epitopes

  • Membrane association may limit antibody access, particularly for C-terminal epitopes

  • Different fixation/permeabilization protocols significantly impact epitope exposure

• Phosphorylation-dependent recognition:

  • Antibodies may have differential affinity for phosphorylated vs. non-phosphorylated forms

  • TBK1 activity varies across cell types and conditions, affecting STX17 phosphorylation

• Expression level considerations:

  • Endogenous STX17 levels can be relatively low in some cell types

  • Current commercial antibodies may lack sensitivity for detecting low expression

• Technical variables:

  • Batch-to-batch variation in antibody production

  • Buffer composition effects on antibody performance

  • Protein extraction method influences membrane protein recovery

To address these challenges, comprehensive validation across intended applications, inclusion of appropriate controls (STX17 knockout/knockdown), and optimization of protocols for specific experimental systems are strongly recommended.

What are the recommended controls when studying STX17 phosphorylation in autophagy research?

When investigating STX17 phosphorylation in autophagy research, implement these essential controls:

• Phosphatase treatment controls:

  • λ phosphatase treatment of samples serves as a negative control for phospho-specific antibodies

  • Include both treated and untreated samples from the same experimental condition

• Genetic controls:

  • TBK1 knockout or knockdown cells (primary kinase for S202 phosphorylation)

  • STX17 knockout cells as absolute negative controls

  • Phospho-mutants (S202A) and phosphomimetics (S202D/E) for functional studies

• Autophagy condition controls:

  • Basal vs. starvation-induced autophagy

  • Autophagy inhibition (e.g., with wortmannin or bafilomycin A1)

  • Time-course analysis during autophagy induction

• Technical considerations:

  • Rapid sample processing with phosphatase inhibitors to preserve phosphorylation status

  • Multiple detection methods (Western blot and immunofluorescence)

  • Quantitative analysis with normalization to total STX17 levels

This comprehensive control strategy enables reliable interpretation of phosphorylation-dependent STX17 functions in the context of autophagy regulation .

How can I optimize immunoprecipitation protocols for studying STX17 complexes?

Optimizing immunoprecipitation (IP) of STX17 complexes requires addressing several technical challenges:

• Membrane protein solubilization:

  • Test different detergents: Start with milder options (0.5-1% CHAPS or digitonin) that better preserve protein-protein interactions

  • Avoid harsh detergents like SDS that disrupt SNARE complexes

  • Consider using membrane-permeable crosslinkers (DSP or formaldehyde) before lysis to stabilize transient interactions

• Antibody selection and orientation:

  • Compare multiple antibodies targeting different STX17 epitopes

  • For known complexes, consider reciprocal IPs (e.g., pull down with SNAP29 antibody)

  • Test both direct antibody conjugation to beads and traditional antibody-protein A/G approaches

• Buffer optimization:

  • Include phosphatase inhibitors to preserve phosphorylation states

  • Test different salt concentrations (150-300mM NaCl) to balance specificity and yield

  • Consider including glycerol (5-10%) to stabilize protein structures

• Procedural considerations:

  • Extended incubation times (overnight at 4°C) may improve complex recovery

  • Gentle washing procedures to preserve weaker interactions

  • Elution strategies: compare traditional approaches versus on-bead digestion for mass spectrometry

• Autophagy-specific considerations:

  • Compare different autophagy induction methods (starvation, rapamycin)

  • Include time-course analysis to capture dynamic interactions

  • Consider subcellular fractionation before IP to enrich for autophagosomal membranes

These optimizations have enabled researchers to successfully characterize STX17's interactions with other SNARE proteins and regulatory factors during autophagy .

How do new findings about STX17 recruitment impact our understanding of autophagy regulation?

Recent discoveries regarding STX17 recruitment to autophagosomes represent significant advances in understanding autophagy regulation:

• Temporal safety mechanism: The electrostatic mechanism for STX17 recruitment to mature autophagosomes provides a molecular explanation for how cells prevent premature fusion of lysosomes with unclosed autophagosomes, which could cause harmful leakage of lysosomal enzymes into the cytosol .

• Lipid-based regulation: The finding that PI4P accumulates during autophagosome maturation and mediates STX17 recruitment reveals a new layer of lipid-based regulation in autophagy progression . This complements known phosphoinositide signaling roles (PI3P, PI(3,5)P2) in earlier and later stages.

• Dual functionality: The discovery that STX17 plays roles in both autophagy initiation (via TBK1-mediated phosphorylation) and autophagosome-lysosome fusion indicates its function as a multifaceted regulator across the entire autophagy pathway .

• Evolutionary implications: Conservation of charged residues in STX17 across species suggests evolutionary pressure to maintain this recruitment mechanism, highlighting its fundamental importance in autophagy regulation .

• Therapeutic potential: These insights open new avenues for therapeutic intervention in autophagy-related diseases, potentially through modulation of STX17 recruitment or phosphorylation.

These findings collectively shift our understanding from viewing STX17 as simply an autophagosomal SNARE to recognizing it as a sophisticated sensor that integrates multiple signals to ensure proper autophagy progression .

What is the relationship between STX17 phosphorylation by TBK1 and its role in autophagosome-lysosome fusion?

The relationship between TBK1-mediated phosphorylation of STX17 and its function in autophagosome-lysosome fusion reveals sophisticated regulatory complexity:

• Distinct functional roles: STX17 phosphorylation by TBK1 at S202 primarily controls autophagy initiation through formation of ATG13-containing complexes , while its role in autophagosome-lysosome fusion involves its SNARE domain interacting with SNAP29 and VAMP7/8 .

• Subcellular localization effects: Phosphorylated STX17 (pS202) localizes specifically to the Golgi apparatus , whereas its role in fusion requires recruitment to mature autophagosomes through interactions with negatively charged membrane surfaces .

• Temporal regulation: TBK1-mediated phosphorylation appears to function earlier in the autophagy pathway, while STX17's recruitment to autophagosomes and participation in fusion occur at later stages .

• Potential crosstalk: Though not fully elucidated, phosphorylation status may influence STX17's ability to engage in SNARE complex formation or interact with regulatory proteins.

• Regulatory complexity: The existence of these distinct mechanisms allows for multiple layers of control over STX17's activities, potentially enabling integration of different cellular signals to fine-tune autophagy.

This dual functionality highlights STX17 as a central node in autophagy regulation, with its various activities precisely controlled through spatial segregation, temporal regulation, and post-translational modifications .

What experimental approaches can test the electrostatic model of STX17 recruitment to autophagosomes?

Several experimental approaches can rigorously test the electrostatic model of STX17 recruitment to autophagosomes:

• Mutagenesis studies:

  • Systematic mutation of positively charged residues in STX17's C-terminal region

  • Creation of charge-reversal mutants (positive to negative) to assess recruitment inhibition

  • Introduction of additional positive charges to test for enhanced recruitment

• Membrane charge manipulation:

  • In vitro systems using liposomes with defined phospholipid compositions

  • Enzymatic modification of membrane charge (as demonstrated with Sac1PD to deplete PI4P)

  • Pharmacological manipulation of kinases/phosphatases that regulate phospholipid phosphorylation

• Direct membrane charge measurement:

  • Fluorescent probes that detect membrane surface charge

  • Comparative analysis between early phagophores and mature autophagosomes

• Molecular dynamics simulations:

  • All-atom models of STX17 transmembrane domains

  • Simulation of interaction with membranes of varying lipid composition and charge

• Structural biology approaches:

  • Cryo-electron microscopy of STX17 in membrane environments

  • NMR studies of STX17 transmembrane domain interactions with lipids

These approaches have already provided substantial evidence for the electrostatic model, showing that positively charged STX17 residues interact with increasingly negatively charged autophagosomal membranes during maturation, with PI4P playing a key role in this process .

How should researchers choose between different STX17 antibodies for specific applications?

When selecting STX17 antibodies for specific applications, researchers should consider these key factors:

Additional recommendation: When possible, use antibodies that have been validated by the manufacturer specifically for your application of interest. For critical experiments, consider testing multiple antibodies targeting different epitopes to confirm findings .

What are the comparative advantages of monoclonal versus polyclonal STX17 antibodies?

Understanding the comparative advantages of monoclonal versus polyclonal STX17 antibodies helps researchers make informed selections:

Currently available commercial antibodies include both types, with rabbit polyclonal STX17 antibodies being among the most widely validated for multiple applications . For phospho-specific detection of STX17 pS202, monoclonal antibodies may offer superior specificity .

What emerging techniques might advance our understanding of STX17 dynamics during autophagy?

Several cutting-edge techniques show promise for revolutionizing our understanding of STX17 dynamics during autophagy:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, STED) to visualize STX17 nanoscale distribution

  • Lattice light-sheet microscopy for long-term 3D imaging with minimal phototoxicity

  • Correlative light and electron microscopy (CLEM) to connect fluorescence patterns with ultrastructure

• Single-molecule techniques:

  • Single-molecule tracking to monitor STX17 mobility and clustering

  • Single-molecule pull-down (SiMPull) assays to analyze complex formation stoichiometry

  • Super-resolution microscopy combined with single-particle tracking

• Integrative structural approaches:

  • Cryo-electron tomography of STX17 in native membrane environments

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Integrative modeling combining multiple structural data sources

• Genome engineering advances:

  • Endogenous tagging of STX17 using CRISPR-Cas9 for physiological expression levels

  • Optogenetic control of STX17 recruitment or function

  • Synthetic biology approaches to engineer autophagy pathways with modified STX17

• Systems biology integration:

  • Multi-omics approaches combining proteomics, lipidomics, and functional genomics

  • Mathematical modeling of STX17 dynamics during autophagy progression

  • Machine learning analysis of large-scale imaging and interaction data

These emerging techniques will help address outstanding questions about STX17's precise temporal regulation, its coordination with other autophagy regulators, and its potential functions beyond canonical autophagy pathways .

How might STX17 antibodies contribute to understanding autophagy dysregulation in disease?

STX17 antibodies offer significant potential for elucidating autophagy dysregulation in various diseases:

• Neurodegenerative disorders:

  • Detecting altered STX17 localization or expression in Alzheimer's, Parkinson's, or Huntington's disease models

  • Evaluating changes in phosphorylated STX17 (pS202) as potential biomarkers

  • Assessing autophagosome maturation defects through STX17 recruitment analysis

• Cancer research applications:

  • Investigating STX17 expression patterns across tumor types and correlation with autophagy status

  • Evaluating STX17 phosphorylation as a marker for TBK1 activity in cancers

  • Studying potential roles in tumor suppression or promotion through autophagy modulation

• Infectious disease insights:

  • Monitoring STX17 redistribution during pathogen infection

  • Assessing xenophagy (pathogen-targeted autophagy) efficiency using STX17 markers

  • Identifying pathogen-mediated manipulation of STX17 function

• Metabolic disorders:

  • Evaluating STX17 dynamics in response to metabolic stress

  • Investigating lipophagy regulation through STX17-dependent mechanisms

  • Studying implications in conditions like obesity and diabetes

• Aging-related research:

  • Assessing age-related changes in STX17 expression, phosphorylation, and function

  • Correlating autophagy efficiency with STX17 dynamics during aging

  • Investigating interventions that restore proper STX17 function

These applications depend critically on high-quality STX17 antibodies that can distinguish different populations, phosphorylation states, and subcellular localizations of the protein in complex disease environments.

What are the key methodological challenges in studying STX17's multiple functions in different cellular compartments?

Investigating STX17's diverse functions across cellular compartments presents several methodological challenges:

• Distinguishing STX17 populations:

  • Difficulty separating autophagy-related from secretory pathway pools of STX17

  • Need for compartment-specific markers that co-localize with different STX17 populations

  • Technical limitations in simultaneously tracking multiple pools

• Temporal resolution challenges:

  • STX17 redistributes dynamically during autophagy, requiring time-resolved approaches

  • Difficulty capturing transition states between distinct functional pools

  • Synchronization challenges in studying naturally asynchronous processes

• Functional isolation difficulties:

  • Manipulating STX17 often affects multiple pathways simultaneously

  • Challenge of creating domain-specific mutants that affect only certain functions

  • Compensatory mechanisms (e.g., YKT6) that mask STX17 function in certain contexts

• Technical detection limits:

  • Endogenous STX17 detection can be challenging with existing antibodies

  • Commercial antibody limitations in distinguishing phosphorylated forms

  • Membrane protein analysis challenges in certain applications

• Integration of approaches:

  • Need to combine imaging, biochemical, and genetic approaches

  • Challenges in reconciling data from different methodological platforms

  • Requirement for systems-level analysis to understand network effects

Addressing these challenges requires development of more specific reagents, including compartment-targeted STX17 variants, phospho-specific antibodies with improved sensitivity, and advanced imaging approaches with higher spatiotemporal resolution .