DYNC1LI2 Antibody

What is DYNC1LI2 and what cellular functions does it perform?

DYNC1LI2 (Dynein Cytoplasmic 1 Light Intermediate Chain 2, also known as LIC2) is a non-catalytic accessory component of the cytoplasmic dynein 1 complex. It plays crucial roles in:

• Linking dynein to cargo and adapter proteins that regulate dynein function

• Facilitating retrograde motility of vesicles and organelles along microtubules

• Potentially binding dynein to membranous organelles or chromosomes

The protein is particularly important in vesicular trafficking mechanisms that maintain cellular homeostasis. Recent research has demonstrated that DYNC1LI2 regulates the localization of chaperone-mediated autophagy (CMA) receptor LAMP2A, which is essential for proper lysosomal function and cellular homeostasis .

DYNC1LI2 and DYNC1LI1 are paralogs (similar proteins resulting from gene duplication) with distinct functions:

• Both are light intermediate chains of cytoplasmic dynein 1

• They show different expression patterns and can be differentially regulated in disease states

• In cystinosis (a lysosomal storage disorder), DYNC1LI2 is specifically downregulated while DYNC1LI1 expression remains normal

• Functionally, DYNC1LI2 reconstitution can rescue certain phenotypes in cystinotic cells, while DYNC1LI1 cannot, suggesting non-redundant functions

This functional differentiation makes it crucial to use specific antibodies that can distinguish between these paralogs in research applications.

What are the optimal conditions for DYNC1LI2 antibody use in Western blotting?

For optimal Western blot results with DYNC1LI2 antibodies:

• Antibody dilution: Typically 1:500-1:5000, with most manufacturers recommending:

  • Polyclonal antibodies: 1:500-1:1000

  • Monoclonal antibodies: 1:1000

• Expected molecular weight: DYNC1LI2 typically appears at:

  • Calculated MW: 54 kDa

  • Observed MW: 48-50 kDa or 60-65 kDa (depending on sample preparation)

• Sample types validated: Human tissues (brain, placenta), mouse tissues (testis, brain, colon), and cell lines (HEK293, NCI-H460, SK-OV-3)

• Sample preparation: Standard SDS-PAGE protocols; no special requirements reported

• Detection method: Standard ECL systems are compatible; specific recommendations not provided in available data

Always optimize conditions for your specific experimental system, especially for varying tissue or cell types .

How should DYNC1LI2 antibodies be stored and handled for maximum stability?

Most manufacturers recommend similar storage conditions for DYNC1LI2 antibodies:

• Storage temperature: -20°C is the standard recommendation

• Buffer composition: Typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Stability: Most are stable for one year after shipment if properly stored

• Aliquoting: While some manufacturers suggest aliquoting is unnecessary for -20°C storage , it's generally good practice to aliquot antibodies to avoid repeated freeze-thaw cycles which can degrade antibody quality

• Handling: Allow antibodies to equilibrate to room temperature before opening to prevent condensation

For specific antibodies, always consult the manufacturer's recommendation as formulations may vary slightly between suppliers.

What controls should be included when validating a new DYNC1LI2 antibody?

When validating a new DYNC1LI2 antibody for your research, include these essential controls:

• Positive tissue/cell controls:

  • Human samples: fetal brain, placenta

  • Mouse samples: testis, brain, colon

  • Cell lines: HEK293, NCI-H460, SK-OV-3

• Negative controls:

  • Primary antibody omission

  • Non-expressing or knockout tissues/cells (if available)

  • Cells treated with DYNC1LI2-specific siRNA or shRNA

• Specificity controls:

  • Pre-absorption with immunizing peptide/antigen

  • Testing cross-reactivity with DYNC1LI1 (the paralog)

  • Comparing results with another validated DYNC1LI2 antibody

• Method-specific controls:

  • For IF/ICC: Include cytoskeleton markers to confirm expected subcellular localization

  • For WB: molecular weight markers and loading controls

This comprehensive validation approach ensures reliable antibody performance across different experimental applications.

How can DYNC1LI2 antibodies be used to study vesicular trafficking defects in disease models?

DYNC1LI2 antibodies have been instrumental in elucidating vesicular trafficking mechanisms, particularly in disease models like cystinosis:

• Co-localization studies: Combine DYNC1LI2 antibodies with markers for:

  • Lysosomes (LAMP1)

  • Chaperone-mediated autophagy receptor (LAMP2A)

  • Endosomal compartments (RAB7, RAB11)

• Live-cell imaging: Use in combination with:

  • LysoTracker for endolysosomal dynamics

  • Total Internal Reflection Fluorescence microscopy (TIRFM) to assess vesicle movement speed and dynamics

• Rescue experiments: Compare trafficking in:

  • Wild-type cells

  • Cells with DYNC1LI2 downregulation (using shRNA)

  • Disease model cells (e.g., cystinotic cells)

  • Disease cells with reconstituted DYNC1LI2 expression

• Functional assays: Measure:

  • Chaperone-mediated autophagy (CMA) activity

  • Endoplasmic reticulum stress markers (HSPA5/GRP78, ATF4, DDIT3/CHOP)

  • Mitochondrial dynamics and function

These approaches have revealed that DYNC1LI2 is essential for proper vesicular trafficking, and its dysfunction contributes to disease pathology in lysosomal storage disorders .

What strategies can address cross-reactivity or background issues with DYNC1LI2 antibodies?

When facing cross-reactivity or high background with DYNC1LI2 antibodies, implement these research-validated solutions:

• Antibody selection strategies:

  • Choose antibodies raised against unique regions that differentiate DYNC1LI2 from its paralog DYNC1LI1

  • Monoclonal antibodies (like EPR11230(2)) may offer higher specificity than polyclonals for certain applications

  • Consider the immunogen used - antibodies generated against recombinant fragments from non-conserved regions tend to show less cross-reactivity

• Protocol optimization:

  • Increase blocking time/concentration (5% BSA or milk is typically effective)

  • Optimize primary antibody concentration with titration experiments

  • Increase washing steps duration and number

  • For IHC, test different antigen retrieval methods (both TE buffer pH 9.0 and citrate buffer pH 6.0 have been used successfully)

• Advanced validation approaches:

  • Perform parallel experiments with DYNC1LI2 knockdown/knockout samples

  • Use peptide competition assays with the immunizing peptide

  • Compare results across multiple DYNC1LI2 antibodies targeting different epitopes

• Application-specific strategies:

  • For IF: Pre-absorb secondary antibodies with tissue powder from the species being studied

  • For WB: Use gradient gels to better separate proteins of similar molecular weight

These strategies help ensure specific detection of DYNC1LI2 and minimize artifacts that could complicate data interpretation.

How can researchers study the interaction between DYNC1LI2 and other components of the dynein complex?

To investigate DYNC1LI2's interactions within the dynein complex and with cargo proteins:

• Co-immunoprecipitation (Co-IP) approaches:

  • Use DYNC1LI2 antibodies to pull down the protein and associated complex members

  • Alternatively, pull down other dynein components and probe for DYNC1LI2

  • Validate results with reciprocal Co-IPs to confirm specific interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusion proteins to identify proximal interactors in living cells

  • For DYNC1LI2-specific interactions compared to DYNC1LI1, parallel experiments with both proteins are informative

• Subcellular fractionation:

  • Combine with immunoblotting to determine compartment-specific interactions

  • Particularly useful for studying association with membranous organelles or chromosomes

• Functional interaction studies:

  • Co-express DYNC1LI2 with dominant-negative versions of potential interactors (e.g., DN-RAB7 or DN-RAB11)

  • Assess effects on trafficking, CMA activity, and cellular homeostasis

  • Compare with DYNC1LI1 to identify paralog-specific interactions

• Structural approaches:

  • Cross-linking mass spectrometry to map interaction interfaces

  • Cryo-EM to visualize DYNC1LI2 within the dynein complex

These methodologies have revealed that DYNC1LI2 interacts specifically with RAB GTPases and is essential for proper LAMP2A trafficking and chaperone-mediated autophagy .

Why might DYNC1LI2 appear at different molecular weights in Western blot experiments?

Researchers frequently observe variation in DYNC1LI2's apparent molecular weight:

• Expected variation:

  • Calculated MW: 54.1 kDa

  • Observed MW ranges: 48-50 kDa or 60-65 kDa

• Biological factors explaining differences:

  • Post-translational modifications (phosphorylation is common in dynein components)

  • Alternative splicing (multiple isoforms may exist)

  • Tissue-specific modifications (e.g., differences between brain and testis samples)

• Technical factors:

  • Gel percentage affecting migration pattern

  • Buffer systems and running conditions

  • Sample preparation methods (denaturing conditions, heating time)

  • Protein standards used for calibration

• Verification approaches:

  • Compare results with multiple DYNC1LI2 antibodies targeting different epitopes

  • Use recombinant DYNC1LI2 as a reference standard

  • Perform mass spectrometry to confirm protein identity

  • Test samples from DYNC1LI2 knockout or knockdown models as negative controls

Researchers should be aware of these variations when interpreting Western blot results and include appropriate controls to validate band identity.

How can researchers differentiate between specific and non-specific staining in DYNC1LI2 immunohistochemistry?

To distinguish specific from non-specific staining in DYNC1LI2 immunohistochemistry:

• Essential controls:

  • No primary antibody control to assess secondary antibody background

  • Isotype control at matching concentration to primary antibody

  • DYNC1LI2 knockdown or knockout tissue (if available)

  • Pre-absorption with immunizing peptide to confirm specificity

• Tissue-specific considerations:

  • DYNC1LI2 shows specific staining patterns in:

    • Mouse brain tissue

    • Human placenta tissue

    • Rat kidney tissue

  • Compare your staining pattern with these validated tissues

• Optimization strategies:

  • Antigen retrieval: Both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been successfully used

  • Antibody titration: Test multiple dilutions (recommended range: 1:50-1:500)

  • Blocking optimization: Extend blocking time or use different blocking reagents

  • Signal amplification systems: Choose appropriate detection method based on expression level

• Pattern validation:

  • Compare subcellular localization with known DYNC1LI2 distribution (cytoplasmic, with enrichment at microtubule-associated structures)

  • Confirm staining pattern with multiple DYNC1LI2 antibodies targeting different epitopes

These approaches help ensure that the observed staining accurately reflects DYNC1LI2 distribution rather than artifacts.

What are the implications of DYNC1LI2 downregulation in disease models for antibody-based studies?

Studies showing DYNC1LI2 downregulation in disease contexts present both challenges and opportunities for antibody-based research:

• Research implications:

  • DYNC1LI2 is specifically downregulated in cystinosis (a lysosomal storage disorder) while its paralog DYNC1LI1 remains normally expressed

  • This selective downregulation affects vesicular trafficking, chaperone-mediated autophagy, and cellular homeostasis

  • DYNC1LI2 reconstitution can rescue these defects, suggesting therapeutic potential

• Methodological considerations:

  • When studying disease models, antibody sensitivity becomes crucial due to reduced target expression

  • Signal amplification may be necessary for detection in downregulated states

  • Quantitative approaches (western blot densitometry, quantitative IF) require careful standardization

  • Higher antibody concentrations may be needed but can increase background

• Experimental design strategies:

  • Include both wild-type and disease model samples for direct comparison

  • Use multiple detection methods to confirm downregulation (qPCR, western blot, immunofluorescence)

  • Consider parallel analysis of DYNC1LI1 as an internal control

  • Employ rescue experiments (DYNC1LI2 reconstitution) to confirm specificity of observed phenotypes

• Therapeutic research applications:

  • DYNC1LI2 antibodies can assess whether potential therapeutics restore normal expression levels

  • Monitor changes in DYNC1LI2 localization as a marker of therapeutic efficacy

  • Use in combination with functional assays (vesicular trafficking, CMA activity) to correlate expression with function

This disease-specific downregulation makes DYNC1LI2 antibodies valuable tools for both basic research and therapeutic development in lysosomal storage disorders.

How can DYNC1LI2 antibodies contribute to research on chaperone-mediated autophagy (CMA)?

Recent research has established DYNC1LI2's role in regulating chaperone-mediated autophagy, opening new applications for DYNC1LI2 antibodies:

• Co-localization studies:

  • DYNC1LI2 antibodies can be used with CMA receptor LAMP2A antibodies to study their spatial relationship

  • In cystinotic cells, DYNC1LI2 reconstitution rescues the mislocalization of LAMP2A and restores its colocalization with LAMP1-positive lysosomes

  • This approach helps visualize trafficking defects in disease models

• Functional CMA assays:

  • After manipulating DYNC1LI2 levels (overexpression, knockdown), CMA activity can be assessed using:

    • Degradation of recombinant GAPDH (a validated CMA substrate)

    • Detection of CMA substrate translocation under ATP-regenerating conditions

    • Lysosomal isolation followed by in vitro CMA assays

  • DYNC1LI2 antibodies confirm expression levels in these experimental systems

• Therapeutic monitoring:

  • In disorders with CMA defects (like cystinosis), DYNC1LI2 antibodies can monitor:

    • Changes in expression levels following treatment

    • Alterations in subcellular localization

    • Association with trafficking components (RAB7, RAB11)

• Mechanistic studies:

  • DYNC1LI2 antibodies help elucidate the molecular pathway connecting dynein function to CMA by:

    • Identifying interacting partners in immunoprecipitation studies

    • Tracking dynamic changes during cellular stress responses

    • Comparing with DYNC1LI1, which does not rescue CMA defects

These applications highlight the value of DYNC1LI2 antibodies in advancing our understanding of selective autophagy mechanisms.

What considerations are important when designing studies to investigate DYNC1LI2's role in cellular stress responses?

When investigating DYNC1LI2's involvement in cellular stress responses:

• Stress model selection:

  • Oxidative stress: DYNC1LI2 reconstitution increases survival of cystinotic cells under oxidative stress conditions

  • ER stress: DYNC1LI2 expression decreases ER stress markers (HSPA5/GRP78, ATF4, DDIT3/CHOP)

  • Mitochondrial stress: DYNC1LI2 affects mitochondrial fragmentation and membrane potential

• Experimental design considerations:

  • Time course experiments are essential as stress responses are dynamic

  • Dose-response studies help identify threshold effects

  • Combined stressors may reveal synergistic roles

  • Recovery periods after stress removal can demonstrate reversibility

• Analytical approaches:

  • Immunofluorescence: Track DYNC1LI2 redistribution during stress responses

  • Western blotting: Quantify changes in expression levels and post-translational modifications

  • Co-immunoprecipitation: Identify stress-specific interaction partners

  • Live-cell imaging: Monitor dynamic changes in real-time

• Validation strategies:

  • Compare DYNC1LI2 and DYNC1LI1 responses to determine paralog-specific functions

  • Use DYNC1LI2 knockdown and rescue approaches to confirm specificity

  • Employ multiple stress inducers to identify common vs. stressor-specific responses

  • Correlate DYNC1LI2 changes with functional outcomes (cell viability, organelle function)

This research approach has revealed that DYNC1LI2 plays a protective role during cellular stress, with implications for diseases characterized by increased ER stress and oxidative damage.

How do recent findings about DYNC1LI2 inform antibody selection for studying ciliopathies and intraflagellar transport?

While DYNC1LI2 is a component of cytoplasmic dynein-1, recent research exploring dynein complexes provides insights for studying ciliopathies:

• Dynein complex specificity:

  • DYNC1LI2 antibodies target cytoplasmic dynein-1 components

  • For studying ciliopathies, researchers should note the distinction between dynein-1 (cytoplasmic transport) and dynein-2 (intraflagellar transport)

  • Antibody selection should account for this specificity when studying different dynein complexes

• Subunit composition considerations:

  • Research indicates that some subunits are shared between dynein complexes while others are specific

  • TCTEX1D2 associates with dynein-2 but not dynein-1, while Tctex-1 associates with dynein-1

  • This differential composition influences antibody selection for specific research questions

• Technical challenges:

  • Limited availability of antibodies for certain dynein-2 subunits has been noted in the literature

  • When studying related components, validation with multiple approaches becomes crucial

  • For comprehensive dynein complex studies, combining antibodies targeting different subunits provides more complete information

• Experimental design approaches:

  • Immunoprecipitation with DYNC1LI2 antibodies followed by mass spectrometry can identify associated proteins

  • Comparative analysis between wild-type samples and ciliopathy models may reveal altered associations

  • Co-localization studies at the ciliary base can demonstrate functional relationships

These considerations help researchers select appropriate antibodies for studying the specific dynein complexes relevant to their research questions about ciliopathies and intraflagellar transport.

What emerging technologies might enhance the utility of DYNC1LI2 antibodies in future research?

Several technological advances are poised to expand DYNC1LI2 antibody applications:

• Advanced imaging approaches:

  • Super-resolution microscopy techniques (STORM, PALM, SIM) can reveal nanoscale spatial relationships between DYNC1LI2 and other dynein components

  • Live-cell single-molecule tracking could visualize DYNC1LI2 dynamics during vesicular transport

  • FRET-based approaches with labeled antibodies or nanobodies could detect protein-protein interactions in real-time

• Proteomics integration:

  • Coupling DYNC1LI2 immunoprecipitation with mass spectrometry for comprehensive interactome analysis

  • Cross-linking mass spectrometry to map interaction domains within dynein complexes

  • Thermal proximity coaggregation (TPCA) for detecting protein complex formation under various conditions

• Nanobody and recombinant antibody technologies:

  • Development of DYNC1LI2-specific nanobodies for live-cell applications

  • Bi-specific antibodies targeting DYNC1LI2 and cargo molecules to study specific interactions

  • Engineered antibodies with reduced background for improved signal-to-noise ratios

• Spatial biology approaches:

  • Spatial transcriptomics combined with DYNC1LI2 immunostaining to correlate protein localization with gene expression patterns

  • Multiplexed antibody staining using cyclic immunofluorescence or mass cytometry to analyze DYNC1LI2 in the context of multiple cellular markers

  • In situ proximity ligation assays to visualize DYNC1LI2 interactions in fixed tissues

These technological advances will enable more detailed studies of DYNC1LI2's dynamic behavior and context-specific functions in healthy and diseased states.

What are the most pressing questions regarding DYNC1LI2 function that antibody-based research could address?

Several critical knowledge gaps about DYNC1LI2 could be addressed using antibody-based approaches:

• Cargo specificity mechanisms:

  • How does DYNC1LI2 differentiate between various cargos?

  • What molecular determinants govern DYNC1LI2's preference for certain membrane-bound organelles?

  • How do post-translational modifications of DYNC1LI2 affect cargo selection?

• Paralog-specific functions:

  • Why can DYNC1LI2, but not DYNC1LI1, rescue defects in cystinotic cells?

  • What structural features determine these functional differences?

  • Are there tissue-specific roles for each paralog?

• Disease-specific alterations:

  • Beyond cystinosis, how is DYNC1LI2 expression or function altered in other diseases?

  • Could DYNC1LI2 dysregulation contribute to neurodegenerative disorders given dynein's importance in neurons?

  • What mechanisms cause DYNC1LI2 downregulation in disease states?

• Regulatory mechanisms:

  • How is DYNC1LI2 expression regulated at transcriptional and post-transcriptional levels?

  • What signaling pathways modulate DYNC1LI2 function?

  • How do cells balance DYNC1LI1 vs. DYNC1LI2 expression?

• Therapeutic potential:

  • Could targeting DYNC1LI2 expression or function offer therapeutic benefits in diseases with trafficking defects?

  • What approaches might selectively modulate DYNC1LI2 without affecting DYNC1LI1?

  • How does DYNC1LI2 reconstitution improve cellular function in disease models?

Addressing these questions with well-validated antibodies would significantly advance our understanding of cellular trafficking mechanisms and potentially reveal new therapeutic targets.

What protocols yield optimal results when using DYNC1LI2 antibodies for immunofluorescence studies?

For successful immunofluorescence studies with DYNC1LI2 antibodies:

• Sample preparation optimization:

  • Fixation: 4% paraformaldehyde (PFA) fixation for 15-20 minutes yields good results

  • Permeabilization: Triton X-100 (0.1-0.5%) effectively permeabilizes cells for DYNC1LI2 detection

  • Blocking: 5% normal serum (matching secondary antibody host) or BSA for 1 hour at room temperature reduces background

• Antibody application:

  • Primary antibody: Multiple DYNC1LI2 antibodies have been validated for IF, with optimal concentrations around 4 μg/mL

  • Incubation: Overnight at 4°C generally yields optimal signal-to-noise ratio

  • Secondary antibody: Highly cross-adsorbed versions minimize cross-reactivity

• Signal optimization strategies:

  • Increasing antibody concentration may enhance detection of DYNC1LI2 in cells with low expression

  • Extended washing steps (4-6 washes of 5-10 minutes each) help reduce background

  • Using mounting media with anti-fade agents preserves signal during imaging

• Co-staining considerations:

  • Combine DYNC1LI2 staining with microtubule markers to visualize association with cytoskeleton

  • Co-staining with organelle markers (LAMP1, LAMP2A, RAB7, RAB11) helps visualize DYNC1LI2's role in trafficking

  • When multiple rabbit antibodies are needed, consider sequential staining with Fab fragments or directly conjugated antibodies

• Controls and validation:

  • Include DYNC1LI2 knockdown cells as negative controls

  • Compare staining patterns between multiple DYNC1LI2 antibodies

  • Validate subcellular localization with GFP-tagged DYNC1LI2 in live cells

These optimized protocols have successfully visualized DYNC1LI2 in multiple cell types, including HEK293 cells and proximal tubule epithelial cells .

How should researchers select between polyclonal and monoclonal DYNC1LI2 antibodies for specific research applications?

When choosing between polyclonal and monoclonal DYNC1LI2 antibodies:

Application-specific recommendations:

• For Western blotting: Both types perform well; monoclonals offer more consistency but polyclonals may provide stronger signals

• For immunofluorescence: Polyclonal antibodies often provide stronger signals in IF applications, but background can be an issue

• For immunohistochemistry: Polyclonal antibodies have been more extensively validated for IHC applications of DYNC1LI2

• For co-localization studies: Monoclonal antibodies may offer advantages when precise localization is required

• For quantitative applications: Monoclonal antibodies provide more consistent results for comparative studies

Select the antibody type that best matches your specific experimental requirements, considering both the technical demands of your application and the biological questions being addressed.

How does the choice of immunogen affect DYNC1LI2 antibody performance?

The immunogen used to generate DYNC1LI2 antibodies significantly impacts their performance characteristics:

• Common immunogen strategies for DYNC1LI2 antibodies:

  • Recombinant fragments: Many antibodies target regions within aa 350-450 or aa 152-180

  • KLH-conjugated synthetic peptides: Used for some polyclonal antibodies

  • Full-length recombinant protein: Less common but provides comprehensive epitope coverage

• Immunogen location affects antibody utility:

  • N-terminal region antibodies: May detect more isoforms if alternative splicing occurs in C-terminal regions

  • Central region antibodies: Often target functional domains involved in cargo binding

  • C-terminal region antibodies: May detect specific post-translational modifications

• Comparison of commercially available antibodies:

  • ab220844: Targets recombinant fragment within aa 350-450

  • 110138: Generated against recombinant protein (Accession Number: NM_006141)

  • HPA057201: Information on specific immunogen not provided in search results

  • 18885-1-AP: Uses DYNC1LI2 fusion protein Ag13452

• Performance implications:

  • Antibodies against conserved regions may cross-react with DYNC1LI1

  • Antibodies targeting unique regions provide better specificity

  • Some epitopes may be masked in native protein conformations or complexes

  • Post-translational modifications near the epitope may affect antibody binding

• Selection guidance:

  • For detecting all forms of DYNC1LI2: Choose antibodies against highly conserved regions

  • For maximum specificity: Select antibodies against unique regions not shared with DYNC1LI1

  • For studying protein complexes: Consider whether the epitope might be obscured in the assembled complex

  • For studying post-translational modifications: Ensure the epitope doesn't contain modification sites

Understanding the immunogen characteristics helps researchers select antibodies most appropriate for their specific research questions and experimental designs.

What evidence supports the reliability of DYNC1LI2 antibodies for detecting endogenous protein levels in tissues versus cell lines?

The reliability of DYNC1LI2 antibodies varies between tissues and cell lines, as supported by the following evidence:

• Tissue validation data:

  • Multiple DYNC1LI2 antibodies have been validated in various tissues including:

    • Human tissues: brain, placenta

    • Mouse tissues: brain, testis, colon, kidney

    • Rat tissues: kidney

  • Consistent detection patterns across multiple antibodies provide confidence in tissue reliability

  • DYNC1LI2 antibodies successfully detected decreased expression in ctns-/- mouse kidney samples compared to wild-type

• Cell line validation:

  • Validated cell lines include:

    • Human cell lines: HEK293, NCI-H460, SK-OV-3

    • Mouse embryonic fibroblasts (MEFs)

    • Proximal tubule cells (PTCs)

  • Antibodies detect both endogenous and overexpressed DYNC1LI2 in these models

• Comparative performance:

  • Antibody performance may vary between tissues and cell lines due to:

    • Expression level differences (typically higher in neural tissues)

    • Processing modifications specific to tissues

    • Background interference levels (often higher in tissues than cell lines)

• Validation through multiple approaches:

  • Western blot detection correlates with mRNA expression levels measured by qPCR

  • Immunofluorescence patterns match the expected subcellular distribution of dynein components

  • Decreased immunoreactivity in knockdown/knockout models confirms specificity

  • Multiple antibodies show consistent expression patterns across tissues and cell lines

• Methodological considerations:

  • Tissues often require more optimization of fixation and antigen retrieval than cell lines

  • Different antibody dilutions may be optimal for tissues (1:50-1:500) versus cell lines (1:500-1:1000)

  • Background may be higher in tissues, requiring more stringent blocking and washing

This evidence demonstrates that properly validated DYNC1LI2 antibodies reliably detect endogenous protein in both tissues and cell lines, though optimal protocols may differ between these sample types.