KIAA0226L Antibody

What is KIAA0226L and what are its alternative names in scientific literature?

KIAA0226L, also known as RUBCNL (Rubicon-like), is a protein that functions as a regulator of autophagy. This protein has several alternative names in scientific literature, including:

• C13orf18

• Protein associated with UVRAG as autophagy enhancer (Pacer)

• RUN and cysteine rich domain containing beclin 1 interacting protein like

• Protein Rubicon-like

In humans, the canonical protein has a reported length of 662 amino acid residues and a mass of 73.5 kDa. Its subcellular localization is primarily in cytoplasmic vesicles and membrane structures . Up to six different isoforms have been reported for this protein, making experimental design considerations important when targeting specific variants .

How does KIAA0226L function in autophagy pathways?

KIAA0226L/RUBCNL functions as a regulator of autophagy that promotes autophagosome maturation by facilitating the biogenesis of phosphatidylinositol 3-phosphate (PtdIns(3)P) in late steps of autophagy . Mechanistically, it acts by:

• Antagonizing RUBCN (Rubicon), thereby stimulating phosphatidylinositol 3-kinase activity of the PI3K/PI3KC3 complex

• Following anchorage to the autophagosomal SNARE STX17, it promotes the recruitment of PI3K/PI3KC3 and HOPS complexes to the autophagosome

• Regulating the fusion specificity of autophagosomes with late endosomes/lysosomes

• Binding phosphoinositides including phosphatidylinositol 3-phosphate (PtdIns(3)P), 4-phosphate (PtdIns(4)P), and 5-phosphate (PtdIns(5)P)

Beyond autophagy, RUBCNL also acts as a regulator of lipid and glycogen homeostasis and may function as a tumor suppressor .

What is the tissue expression pattern of KIAA0226L?

KIAA0226L expression has been detected in various tissues, though expression levels vary significantly. Based on available immunohistochemistry data:

For research purposes, human U-251 MG (brain glioma), RT4 (urinary bladder cancer), HeLa, HepG2, and A431 cell lines have been validated for KIAA0226L expression and can serve as positive controls in various experimental applications .

Which antibody applications are most effective for KIAA0226L detection?

The effectiveness of antibody applications for KIAA0226L/RUBCNL detection varies based on experimental needs:

When selecting an antibody for KIAA0226L detection, consider target epitope location, as different antibodies target different regions of the protein. For example, antibodies targeting amino acids 150-350, 859-972, or the N-terminal region are available with different validation profiles .

How should researchers optimize antibody dilution for KIAA0226L detection?

Optimization of antibody dilution is critical for specific KIAA0226L detection:

• Begin with the manufacturer's recommended dilution range (e.g., 1:500-1:2000 for WB or 1:50-1:500 for IF)

• Perform a dilution series experiment:

  • For Western blot: Test 3-4 dilutions across the recommended range using a positive control sample

  • For IHC/IF: Use a known positive sample and test dilutions in 2-fold or 3-fold increments

• Evaluate signal-to-noise ratio at each dilution

• Consider sample-specific optimization:

  • Cell lines may require different optimal dilutions than tissue samples

  • Fixation methods can affect optimal antibody concentration

For KIAA0226L specifically, note that the observed molecular weight in Western blot ranges from 110-130 kDa , which is higher than the predicted 73.5 kDa , likely due to post-translational modifications.

What are the recommended fixation and permeabilization protocols for immunofluorescence detection of KIAA0226L?

Based on validated protocols for KIAA0226L immunofluorescence:

Recommended protocol:

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

• Wash 3 times with PBS

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes

• Block with 1-5% BSA or serum for 30-60 minutes

• Incubate with primary KIAA0226L antibody (1:50-1:500 dilution) overnight at 4°C

• Wash 3 times with PBS

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Wash, counterstain with DAPI, and mount

For enhanced visualization of KIAA0226L's association with autophagosomal structures, co-staining with markers such as LC3 or STX17 is recommended .

How can researchers study the interaction between KIAA0226L and the PI3K/PI3KC3 complex?

To investigate KIAA0226L's interaction with the PI3K/PI3KC3 complex:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate KIAA0226L using validated antibodies

  • Probe for PI3K/PI3KC3 complex components (BECN1, VPS34, etc.)

  • Alternatively, perform the reverse IP and probe for KIAA0226L

• Proximity Ligation Assay (PLA):

  • Use primary antibodies against KIAA0226L and PI3K components

  • This technique visualizes protein-protein interactions in situ with high specificity

• FRET/BRET Analysis:

  • Generate fluorescent protein-tagged constructs of KIAA0226L and PI3K components

  • Measure energy transfer as an indicator of protein proximity

• Kinase Activity Assays:

  • Compare PI3K activity in the presence and absence of KIAA0226L

  • Measure PtdIns(3)P production using specific detection methods

• Domain Mapping:

  • Generate deletion constructs of KIAA0226L to identify domains critical for PI3K interaction

  • Focus on the middle region that may include a PI3K-binding domain, similar to that found in RUBCN

These approaches can be combined with autophagy induction or inhibition to understand the dynamics of KIAA0226L-PI3K interaction during autophagy progression.

What methodologies can be employed to investigate KIAA0226L's potential role as a tumor suppressor?

To study KIAA0226L's potential tumor suppressor function:

• Expression Analysis in Cancer Tissues:

  • Compare KIAA0226L expression in tumor vs. normal tissues using IHC with optimized antibody protocols (1:20-1:200 dilution)

  • Correlate expression levels with clinical parameters and patient outcomes

• Loss-of-Function Studies:

  • Generate KIAA0226L knockdown or knockout cell lines using siRNA, shRNA, or CRISPR-Cas9

  • Assess proliferation, migration, invasion, and colony formation

  • Evaluate autophagy flux and its relationship to tumorigenic properties

• Gain-of-Function Studies:

  • Overexpress KIAA0226L in cancer cell lines with low endogenous expression

  • Measure changes in tumorigenic properties and autophagy markers

• In vivo Tumor Models:

  • Implant KIAA0226L-manipulated cancer cells in animal models

  • Monitor tumor growth, metastasis, and response to therapy

• Mechanistic Studies:

  • Investigate KIAA0226L-mediated regulation of oncogenic signaling pathways

  • Identify binding partners specific to tumor suppression functions

  • Examine the relationship between KIAA0226L-regulated autophagy and cancer cell survival

This multifaceted approach can help establish whether KIAA0226L functions as a bona fide tumor suppressor and through what mechanisms it exerts this function.

How can researchers differentiate between RUBCNL (KIAA0226L) and RUBCN (KIAA0226) in experimental systems?

Distinguishing between these related proteins requires careful experimental design:

For definitive differentiation in co-expression systems:

• Use epitope-tagged constructs with different tags (e.g., RUBCNL-GFP and RUBCN-RFP)

• Perform siRNA knockdown specific to each protein and confirm specificity of antibody detection

• In knockout systems, ensure complete removal of one protein does not affect detection of the other

When interpreting results, remember that RUBCNL antagonizes RUBCN in autophagy regulation , so their functions are often opposing rather than redundant.

What are common causes of non-specific binding with KIAA0226L antibodies and how can they be addressed?

Common causes and solutions for non-specific binding:

KIAA0226L-specific considerations:

• The observed molecular weight may be higher (110-130 kDa) than predicted (73.5 kDa) due to post-translational modifications

• When using antibodies that potentially cross-react with RUBCN, include appropriate positive and negative controls

• For challenging applications, consider using monoclonal antibodies with defined epitopes, such as clone 4F5B5 which targets the AA 859-972 region

How can researchers validate the specificity of KIAA0226L antibodies?

Comprehensive validation approaches for KIAA0226L antibodies:

• Genetic Knockdown/Knockout Validation:

  • Perform siRNA/shRNA knockdown or CRISPR knockout of KIAA0226L

  • Compare antibody signal between control and knockdown/knockout samples

  • Signal should be significantly reduced or absent in knockdown/knockout samples

• Overexpression Validation:

  • Transfect cells with KIAA0226L expression constructs

  • Verify increased antibody signal in overexpressing cells

• Peptide Competition Assay:

  • Pre-incubate antibody with immunizing peptide before application

  • Signal should be blocked in the presence of competing peptide

• Multiple Antibody Comparison:

  • Use antibodies targeting different epitopes of KIAA0226L

  • Consistent results with different antibodies suggest specificity

• Molecular Weight Verification:

  • Confirm detected protein size matches expected KIAA0226L size (~73.5 kDa)

  • Note that post-translational modifications may alter the apparent molecular weight (110-130 kDa observed)

For Western blot applications, validated positive controls include U-251 MG, RT4, HeLa, and HepG2 cell lysates . For IHC applications, human duodenum, testis, and tonsillitis tissues have been validated as positive controls .

What controls should be included when studying KIAA0226L in autophagy research?

Essential controls for KIAA0226L autophagy research:

For advanced autophagy studies:

• Include co-staining with established autophagy markers (LC3, p62/SQSTM1, STX17)

• Use tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosome formation from maturation

• Employ live-cell imaging to track KIAA0226L dynamics during autophagy progression

• Include readouts for PI3K activity and PtdIns(3)P production to link KIAA0226L function to its proposed mechanism

When investigating KIAA0226L's antagonistic relationship with RUBCN, include experimental conditions where both proteins are manipulated to demonstrate their opposing functions in autophagy regulation.

What are promising research avenues for understanding KIAA0226L function beyond autophagy?

Emerging areas for KIAA0226L research beyond autophagy:

• Lipid and Glycogen Homeostasis:

  • Investigate KIAA0226L's role in lipid metabolism using lipidomics approaches

  • Examine glycogen synthesis and breakdown in KIAA0226L-deficient models

  • Study metabolic phenotypes in tissue-specific knockout models

• Cancer Biology:

  • Explore KIAA0226L as a biomarker in different cancer types

  • Investigate its tumor suppressor mechanisms independent of autophagy

  • Develop therapeutic approaches targeting KIAA0226L pathways

• Interaction with Other Cellular Pathways:

  • Study KIAA0226L in relation to endocytic pathways

  • Explore connections to mTOR signaling

  • Investigate potential roles in inflammatory responses

• Structural Biology:

  • Determine the three-dimensional structure of KIAA0226L

  • Map functional domains and interaction surfaces

  • Compare structural features with RUBCN to understand their opposing functions

• Developmental Biology:

  • Characterize KIAA0226L expression during development

  • Investigate phenotypes of developmental knockout models

  • Study tissue-specific functions in model organisms

These research directions may benefit from emerging technologies such as proximity labeling, single-cell analysis, and cryo-EM structural determination to provide deeper insights into KIAA0226L biology.

How can researchers study the evolutionary conservation and divergence of KIAA0226L across species?

Methodological approaches for evolutionary studies of KIAA0226L:

• Comparative Genomics:

  • Analyze KIAA0226L orthologs across species (mouse, rat, bovine, frog, zebrafish, chimpanzee, chicken)

  • Identify conserved domains and regulatory regions

  • Construct phylogenetic trees to trace evolutionary history

• Cross-Species Functional Conservation:

  • Test whether orthologs from different species can rescue KIAA0226L deficiency

  • Compare subcellular localization patterns across species

  • Examine species-specific interaction partners

• Domain Evolution Analysis:

  • Map functional domains that are conserved versus divergent

  • Identify species-specific insertions or deletions

  • Correlate domain changes with functional adaptations

• Expression Pattern Comparison:

  • Compare tissue-specific expression across species

  • Identify conserved versus species-specific regulatory elements

  • Link expression differences to physiological adaptations

• Experimental Models:

  • Generate cross-species antibodies that recognize conserved epitopes

  • Develop model organism systems (zebrafish, Drosophila) for functional studies

  • Create domain-swapping experiments between KIAA0226L and RUBCN to understand functional divergence

This evolutionary perspective can provide valuable insights into the core functions of KIAA0226L that have been preserved across species versus lineage-specific adaptations.

What methodological approaches can be used to study the role of KIAA0226L in neurodegenerative diseases?

Methodological framework for investigating KIAA0226L in neurodegeneration:

• Expression Analysis in Disease Models:

  • Examine KIAA0226L levels in post-mortem brain tissues from patients with neurodegenerative diseases

  • Analyze expression in cellular and animal models of neurodegeneration

  • Use validated antibodies optimized for neural tissue (IHC dilution 1:20-1:200)

• Functional Studies in Neuronal Models:

  • Generate KIAA0226L knockdown/knockout in neuronal cell lines and primary neurons

  • Assess effects on neuronal autophagy, protein aggregation, and cell survival

  • Use live imaging to track autophagosome formation and maturation in neurons

• Animal Model Studies:

  • Create neuron-specific KIAA0226L knockout models

  • Examine behavioral, histological, and biochemical phenotypes

  • Test whether KIAA0226L modulation affects disease progression in existing neurodegeneration models

• Mechanistic Investigations:

  • Study KIAA0226L interactions with disease-relevant proteins (tau, α-synuclein, Aβ)

  • Examine the role of KIAA0226L in clearing protein aggregates via autophagy

  • Investigate KIAA0226L regulation of neuroinflammation

• Therapeutic Potential:

  • Develop approaches to modulate KIAA0226L activity in neurons

  • Test whether enhancing KIAA0226L function promotes autophagic clearance of aggregates

  • Evaluate KIAA0226L as a biomarker for disease progression or treatment response