TBC1D14 Antibody

What is TBC1D14 and what cellular functions does it regulate?

TBC1D14 (TBC1 Domain Family, Member 14) is a protein that functions as a negative regulator of autophagy. It acts primarily by controlling membrane delivery from RAB11-positive recycling endosomes to forming autophagosomes . TBC1D14 contains a TBC domain at its C-terminus (residues 411-611), which is characteristic of proteins with potential GTPase-activating protein (GAP) activity for Rab family proteins .

The protein has been shown to interact with the TRAPP (trafficking protein particle) complex via its N-terminal region (amino acids 120-223), also known as the TRAPP-binding region (TBR) . This interaction is crucial for regulating both autophagic and secretory pathways, as TBC1D14 appears to function as a bridge between a TRAPP complex and activated RAB11 . Importantly, while TBC1D14 can bind to RAB11, it may not act as a GAP for RAB11 but rather as an effector .

Beyond its role in autophagy, recent research has implicated TBC1D14 in cancer progression. Studies have shown that TBC1D14 has lower expression in head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis and may serve as a favorable prognostic indicator .

What epitopes are targeted by common TBC1D14 antibodies and why does this matter?

Commercial TBC1D14 antibodies target various regions of the protein, with significant functional and experimental implications. The choice of epitope can dramatically affect experimental outcomes based on the protein domains and interactions being studied.

When selecting a TBC1D14 antibody, researchers should consider which functional domain they're investigating. For studying TRAPP complex interactions, antibodies targeting the N-terminal region may be more informative, while those investigating potential GAP activity might prefer antibodies targeting the C-terminal TBC domain .

How can researchers validate TBC1D14 antibody specificity in their experimental systems?

Validating TBC1D14 antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation approach should include:

• Western blot analysis with positive and negative controls:

  • Use lysates from cells known to express TBC1D14 versus knockout/knockdown cells

  • Look for a single band at the expected molecular weight (~78 kDa)

  • Include species-appropriate controls based on the antibody's reactivity profile (human and mouse for ABIN955090)

• Immunoprecipitation validation:

  • Perform IP with the TBC1D14 antibody followed by mass spectrometry analysis

  • Confirm pulled-down proteins include TBC1D14 and known interactors like TRAPP complex subunits

  • Compare results with those from GST pull-down assays as demonstrated in previous studies

• Immunofluorescence and colocalization:

  • Validate subcellular localization by comparing with known TBC1D14 patterns (recycling endosomes)

  • Perform co-staining with RAB11 (should show significant colocalization)

  • Upon overexpression, verify formation of tubulated transferrin-positive recycling endosome compartments

• Genetic approaches:

  • Use siRNA knockdown of TBC1D14 and show reduced antibody signal

  • For advanced validation, employ CRISPR-Cas9 knockout cells as negative controls

How can TBC1D14 antibodies be utilized to study protein's role in autophagy regulation?

Studying TBC1D14's role in autophagy regulation requires sophisticated approaches leveraging specific antibodies:

• Monitoring autophagy inhibition:

  • Use TBC1D14 antibodies to track protein expression/localization during autophagy induction

  • Correlate TBC1D14 levels with LC3 lipidation (LC3-I to LC3-II conversion) by immunoblotting

  • Previous studies have shown that TBC1D14 overexpression reduces LC3 lipidation, indicating autophagy inhibition

• Visualizing recycling endosome-autophagosome interactions:

  • Perform triple immunofluorescence using antibodies against TBC1D14, RAB11 (recycling endosome marker), and autophagy proteins (ULK1, ATG9, LC3)

  • This approach reveals how TBC1D14 mediates membrane trafficking from recycling endosomes to forming autophagosomes

  • Time-lapse imaging with fluorescently tagged proteins can complement antibody-based fixed-cell approaches

• Analyzing TBC1D14-TRAPP complex interactions during autophagy:

  • Use co-immunoprecipitation with TBC1D14 antibodies under basal and starvation conditions

  • Probe for TRAPP complex components (particularly TRAPPC8, TRAPPC4, and TRAPPC12)

  • Quantify changes in interaction during autophagy activation

• Proximity labeling approaches:

  • Combine TBC1D14 antibodies with BioID-based approaches as demonstrated in previous research

  • This allows identification of proteins in close proximity to TBC1D14 under different conditions

  • Previous studies identified TRAPPC8 as most proximal to TBC1D14 using this approach

What are the optimal protocols for analyzing TBC1D14's interactions with the TRAPP complex?

Analysis of TBC1D14-TRAPP interactions requires tailored methodological approaches:

• Co-immunoprecipitation (Co-IP) protocol:

  • Lyse cells in buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and protease inhibitors

  • Incubate cell lysate with TBC1D14 antibody (e.g., ABIN955090) overnight at 4°C

  • Add protein A/G beads and incubate for 2-3 hours

  • Wash with lysis buffer and elute with SDS sample buffer

  • Analyze by immunoblotting for TRAPP complex subunits (TRAPPC4, TRAPPC8, TRAPPC12)

• GST pull-down assay:

  • Express and purify GST-tagged TBC1D14 (full length or TBR domain, aa 120-223)

  • Incubate with cell lysate containing TRAPP complex components

  • Wash and elute bound proteins for mass spectrometry analysis

  • This approach successfully identified multiple TRAPP components in previous studies

• Proximity labeling with BioID:

  • Generate myc-BioID-TBC1D14 constructs

  • Express in cells and add biotin to culture medium

  • Perform streptavidin pull-downs under denaturing conditions (1% SDS)

  • Analyze biotinylated proteins by mass spectrometry

  • This approach identified TRAPPC8 as the most proximal subunit to TBC1D14

• Immunofluorescence co-localization:

  • Transfect cells with GFP-TBC1D14 and perform immunostaining for TRAPP components

  • Focus on co-localization with TRAPPC4 on tubulated endosomes

  • Analyze RAB1 and RAB11 co-localization on these structures

  • Use super-resolution microscopy for detailed analysis of protein proximity

How do different TBC1D14 domains contribute to its function, and how can domain-specific antibodies help elucidate this?

TBC1D14's function is determined by distinct domains with unique roles that can be studied using domain-specific antibodies:

• N-terminal TRAPP-binding region (TBR, aa 120-223):

  • Mediates interaction with TRAPP complex components

  • Overexpression of this region alone inhibits both autophagy and secretory traffic

  • Domain-specific antibodies targeting this region can help track TRAPP complex binding without affecting RAB11 interaction

• ULK1-binding region (aa 224-330):

  • Mediates interaction with ULK1 kinase, a core autophagy initiator

  • Domain-specific antibodies can help monitor this interaction during autophagy induction

  • Distinct from the TRAPP-binding region, allowing independent regulation

• TBC domain (aa 411-611):

  • Potentially mediates GAP activity for Rab proteins

  • Structure has been solved (residues 357-672)

  • Does not bind to TRAPP complex components

When using domain-specific antibodies, researchers should consider:

What are the key considerations when designing experiments to study TBC1D14's role in HNSCC progression and lymph node metastasis?

Recent research has implicated TBC1D14 in cancer progression, particularly in head and neck squamous cell carcinoma (HNSCC). When studying this aspect, researchers should consider:

• Expression analysis in clinical samples:

  • Use TBC1D14 antibodies for immunohistochemistry on HNSCC tissue microarrays

  • Compare expression between primary tumors with and without lymph node metastasis

  • Quantify expression using digital pathology algorithms

  • Previous studies have shown lower TBC1D14 expression in HNSCC with lymph node metastasis

• Functional studies in HNSCC cell lines:

  • Design gain/loss-of-function experiments (overexpression/knockdown)

  • Assess effects on:

    • Autophagy (LC3 puncta formation, LC3-II/I ratio)

    • Cell migration (wound healing assays)

    • Invasion (Transwell assays)

    • Interaction with downstream targets (e.g., MAEA)

• In vivo metastasis models:

  • Develop TBC1D14-modulated HNSCC cell lines

  • Implant in appropriate animal models

  • Monitor lymph node metastasis formation

  • Perform immunohistochemistry on primary tumors and metastases

• Correlation with autophagy markers:

  • Co-stain for TBC1D14 and autophagy markers (LC3, p62/SQSTM1)

  • Analyze correlation between TBC1D14 levels, autophagy activity, and metastatic potential

  • Previous research indicates TBC1D14 inhibits autophagy to suppress lymph node metastasis

How can researchers optimize TBC1D14 antibody use in multicolor immunofluorescence for studying recycling endosome tubulation?

TBC1D14 overexpression generates distinctive tubulated endosomal structures that can be visualized using optimized immunofluorescence techniques:

• Sample preparation protocol:

  • Transfect cells with GFP-TBC1D14 (or use endogenous TBC1D14 with appropriate antibodies)

  • Fix with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1% Triton X-100 for 5 minutes

  • Block with 5% normal serum in PBS for 1 hour

• Antibody combinations for optimal visualization:

  • Primary antibody set 1: TBC1D14 + RAB11 + RAB1B

    • This combination reveals how TBC1D14 tubules can simultaneously harbor both RAB11 and RAB1

  • Primary antibody set 2: TBC1D14 + TRAPPC4 + RAB11

    • Demonstrates co-localization of TRAPPC4 and RAB11 on TBC1D14 tubules

  • Primary antibody set 3: TBC1D14 + TRAPPC4 + RAB1B

    • Shows TRAPPC4 and RAB1B co-localization on tubules

• Advanced imaging considerations:

  • Use confocal microscopy with high NA objectives (≥1.3) for optimal resolution

  • Consider super-resolution approaches (STED, SIM, STORM) for detailed tubule morphology

  • Perform live-cell imaging with fluorescently tagged proteins to observe tubule dynamics

  • Take Z-stacks (0.3μm steps) to capture the full 3D structure of tubules

• Quantitative analysis:

  • Measure tubule length, number per cell, and branching pattern

  • Quantify co-localization coefficients between TBC1D14 and various markers

  • Compare tubulation under different conditions (starvation, drug treatments)

What experimental controls are essential when using TBC1D14 antibodies for various applications?

Rigorous experimental controls are critical for generating reliable data with TBC1D14 antibodies:

• Western blotting controls:

  • Positive control: Lysate from cells with confirmed TBC1D14 expression

  • Negative control: Lysate from TBC1D14 knockdown/knockout cells

  • Loading control: Housekeeping protein (e.g., β-actin, GAPDH)

  • Molecular weight marker: Verify the expected 78 kDa band

  • Secondary antibody-only control: Check for non-specific binding

• Immunoprecipitation controls:

  • IgG control: Non-specific IgG from the same species as the TBC1D14 antibody

  • Input control: Analyze a portion of the lysate before IP

  • Specificity control: Perform IP with and without TBC1D14 overexpression

• Immunofluorescence controls:

  • Primary antibody omission: Detect secondary antibody non-specific binding

  • Blocking peptide competition: Confirm epitope specificity

  • Knockdown validation: Reduced signal in TBC1D14-depleted cells

  • For tubulation experiments: Compare with RAB11 staining patterns

• Function-blocking experiments:

  • When using antibodies to disrupt TBC1D14 function, include:

    • Isotype control antibodies

    • Dose-response curves

    • Timing controls (pre-incubation, co-incubation)

What are the challenges in detecting endogenous TBC1D14 and how can they be overcome?

Detecting endogenous TBC1D14 presents several challenges that can be addressed with specialized approaches:

• Low expression levels:

  • Challenge: TBC1D14 may be expressed at low levels in some cell types

  • Solution: Use signal amplification methods like TSA (tyramide signal amplification) for immunofluorescence

  • Alternative: Concentrate proteins by immunoprecipitation before Western blotting

• Antibody specificity issues:

  • Challenge: Cross-reactivity with related TBC domain proteins

  • Solution: Validate with genetic approaches (siRNA, CRISPR knockout)

  • Alternative: Use multiple antibodies targeting different epitopes

• Subcellular localization:

  • Challenge: Detecting dispersed protein across recycling endosomes

  • Solution: Use super-resolution microscopy techniques

  • Alternative: Employ subcellular fractionation followed by Western blotting

• Dynamic regulation during autophagy:

  • Challenge: Capturing temporal changes in localization

  • Solution: Use synchronized cell populations with timed autophagy induction

  • Alternative: Live-cell imaging with knock-in fluorescent tags (CRISPR knock-in)

How can researchers effectively combine TBC1D14 antibodies with proximity labeling techniques to identify novel interaction partners?

Combining TBC1D14 antibodies with proximity labeling offers powerful insights into its interaction network:

• BioID approach optimization:

  • Generate myc-BioID-TBC1D14 fusion constructs

  • Express in target cells and provide biotin (50 μM) for 24 hours

  • Perform streptavidin pull-downs under denaturing conditions (1% SDS)

  • Analyze bound proteins by mass spectrometry

  • Previous studies identified TRAPPC8 as most proximal to TBC1D14 using this approach

• APEX2 proximity labeling:

  • Create APEX2-TBC1D14 fusion proteins

  • Optimize H₂O₂ treatment (1 mM, 1 minute) for biotinylation

  • Capture labeled proteins under various conditions (basal, starvation)

  • Compare interactomes during different cellular states

• Verification with conventional approaches:

  • Confirm key interactions using TBC1D14 antibodies for co-immunoprecipitation

  • Perform reciprocal IPs with antibodies against identified partners

  • Use immunofluorescence to confirm co-localization of TBC1D14 and partners

• Domain-specific interactome analysis:

  • Create truncation constructs (TBR domain, ULK1-binding region, TBC domain)

  • Perform domain-specific proximity labeling

  • Map interaction sites for different partners

  • Cross-validate with in vitro binding assays

What are common issues when using TBC1D14 antibodies and how can they be resolved?

How can researchers address data inconsistencies when studying TBC1D14 in different experimental systems?

Resolving inconsistencies across experimental systems requires systematic troubleshooting:

• Cell type-specific differences:

  • TBC1D14 function may vary between cell types due to different expression levels of interaction partners

  • Solution: Characterize the expression of key interactors (RAB11, TRAPP components) in each cell system

  • Validate findings across multiple cell lines representing the tissue of interest

• Antibody performance variation:

  • Different antibodies may recognize distinct epitopes/conformations

  • Solution: Test multiple validated antibodies targeting different regions

  • Consider creating a consensus from multiple antibody results

• Technical variability:

  • Fixation methods can affect epitope accessibility in immunofluorescence

  • Solution: Standardize protocols across laboratories

  • Include positive controls with known outcomes in each experiment

• Functional redundancy:

  • Other TBC domain proteins may compensate for TBC1D14

  • Solution: Consider double knockdown experiments

  • Analyze expression of related proteins in different systems