DYNC1LI1 Antibody, Biotin conjugated

What is DYNC1LI1 and what are its cellular functions?

DYNC1LI1 serves as a non-catalytic accessory component of the cytoplasmic dynein 1 complex that links dynein to cargos and adapter proteins regulating dynein function. This protein is integral to intracellular retrograde motility of vesicles and organelles along microtubules . DYNC1LI1 likely binds dynein to membranous organelles or chromosomes and is involved in the microtubule-dependent transport of pericentrin. Research has established that DYNC1LI1 is required for progress through the spindle assembly checkpoint, with its phosphorylated form playing a role in the selective removal of MAD1L1 and MAD1L2 from kinetochores . This protein interacts with numerous partners including REL, BICD2, DYNC1I1, DCTN1, and UBC, indicating its involvement in complex cellular pathways essential for proper cell division and transport.

What experimental applications is the DYNC1LI1 Antibody, Biotin conjugated suitable for?

The DYNC1LI1 Antibody, Biotin conjugated has been validated for multiple research applications including:

• Western Blotting (WB) with confirmed detection of the expected 56kDa band in A549 cell lysates

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunohistochemistry on paraffin-embedded sections (IHC-p)

• Immunohistochemistry on frozen sections (IHC-fro)

The biotin conjugation provides significant advantages for detection sensitivity through streptavidin-based systems, making this antibody particularly useful for detecting lower expression levels of DYNC1LI1 in tissue samples and for signal amplification in various immunoassays.

What is the reactivity profile and technical specifications of the DYNC1LI1 Antibody?

The DYNC1LI1 Antibody demonstrates broad cross-species reactivity, making it valuable for comparative studies:

Technical specifications include:

• Host: Rabbit

• Clonality: Polyclonal

• Immunogen: Synthetic peptide directed towards the C-terminal region (AA 451-523) of human DYNC1LI1

• Purification method: Affinity purified

• Format: Liquid in 1x PBS buffer with 0.09% sodium azide and 2% sucrose

How can researchers validate DYNC1LI1 Antibody specificity in experimental systems?

Comprehensive validation of DYNC1LI1 Antibody requires multiple approaches:

• Western blot validation:

  • Detection of the expected 56kDa band in appropriate cell lysates (e.g., A549 cells)

  • Note that validation data shows multiple additional lower molecular weight bands that could represent degradation products or non-specific binding

• Knockdown validation protocol:

  • Use lentiviral-delivered shRNA targeting DYNC1LI1 (e.g., pLKO_TRC005-DYNC1LI1, clone ID: TRCN0000299843)

  • Compare with control vector (e.g., pLKO_TRC005-luciferase)

  • Confirm knockdown efficiency via RT-qPCR and western blotting after 48 hours of infection

  • Expect significant reduction in the 56kDa band intensity

• Concentration gradient analysis:

  • Test antibody with serial dilutions of cell lysates (e.g., from 500,000 to 25,000 cells)

  • Observe proportional reduction in signal intensity with decreased protein loading

• Cross-reference between techniques:

  • Compare results across WB, IHC, and ELISA for consistent detection patterns

What is the optimal western blotting protocol for DYNC1LI1 Antibody, Biotin conjugated?

Based on validated experimental procedures, the following western blotting protocol is recommended:

Sample preparation:

• Prepare cell lysates from 25,000-500,000 cells using buffer supplemented with protease inhibitors

• Quantify protein using Bio-Rad Protein Assay reagent

• Denature 15 μg protein/lane in NuPAGE LDS sample buffer for 10 min at 95°C

Gel electrophoresis and transfer:

• Separate proteins on 10-12% SDS-PAGE gel

• Transfer to PolyScreen 2 PVDF membrane (0.2 μm)

Antibody incubation:

• Block membrane with 3% bovine serum albumin for 1 hour at room temperature

• Incubate with biotin-conjugated DYNC1LI1 primary antibody at 1:1,000 dilution (optimize empirically)

• Detect using streptavidin-HRP or other streptavidin-coupled detection system

• Include β-actin (1:1,000) as loading control

Important note: The detection of multiple bands below the expected 56kDa molecular weight requires careful interpretation. DYNC1LI1 protein knockdown experiments can help determine if these represent specific degradation products or non-specific binding .

How can shRNA-mediated DYNC1LI1 knockdown be implemented, and what phenotypic changes should researchers expect?

Validated knockdown protocol:

• Construct preparation:

  • Use pLKO_TRC005-DYNC1LI1 (clone ID: TRCN0000299843) containing shRNA targeting DYNC1LI1

  • Prepare pLKO_TRC005-luciferase (clone ID: TRCN0000231719) as negative control

• Transfection procedure:

  • Plate 1.25×10^5 cells/well in a 6-well plate and culture for 24 hours

  • Perform lentiviral infection at MOI=3

  • Select stable clones with 2 mg/ml puromycin

  • Validate knockdown efficiency after 48 hours using RT-qPCR and western blotting

Expected phenotypic changes after DYNC1LI1 knockdown:

These changes suggest DYNC1LI1 knockdown induces a less aggressive cancer phenotype with increased chemosensitivity, demonstrating the value of this approach for investigating DYNC1LI1 function in cancer biology .

How is DYNC1LI1 expression altered in cancer, and what are the implications for research?

DYNC1LI1 shows significant expression alterations across multiple cancer types:

Colorectal cancer (CRC):

• Significantly increased expression in metastatic vs. non-metastatic samples

• LS 174T cells (AJCC stage II) exhibit higher DYNC1LI1 expression compared to other CRC cell lines

Other cancer types:

• Detected in urine of pancreatic ductal adenocarcinoma patients

• Increased phosphorylation observed in prostate cancer

These expression patterns suggest DYNC1LI1 may serve as:

• A potential biomarker for cancer progression and metastasis

• An indicator of chemotherapy sensitivity, particularly to 5-fluorouracil

• A target for functional studies investigating cancer cell behavior

For cancer research applications, it is advisable to include appropriate controls (e.g., LS 174T cells for CRC studies) and correlate DYNC1LI1 expression with clinical parameters and other molecular markers.

What is the relationship between DYNC1LI1 expression and mucin regulation in colorectal cancer?

Research has revealed a significant relationship between DYNC1LI1 and mucin expression in colorectal cancer:

This altered mucin profile has important implications:

• MUC1 overexpression typically correlates with poor prognosis in epithelial cancers

• MUC2 is generally considered protective in colorectal cancer

• The mucin profile after DYNC1LI1 knockdown resembles a less aggressive cancer phenotype

The mechanism linking DYNC1LI1 to mucin regulation remains to be fully elucidated but may involve:

• Direct interactions with transcription factors regulating mucin genes

• Effects on intracellular transport pathways affecting mucin secretion

• Indirect effects through cytoskeletal organization impacting cell differentiation

This relationship positions DYNC1LI1 as a potential upstream regulator of mucosal barrier integrity in cancer, offering new research directions for understanding colorectal cancer biology.

How does DYNC1LI1 expression affect chemosensitivity in cancer cells?

Experimental evidence demonstrates that DYNC1LI1 influences cancer cell response to chemotherapy:

• LS 174T cells with decreased DYNC1LI1 expression show increased sensitivity to 5-fluorouracil (5-FU)

• CRC cells with low MUC1 and high MUC2/MUC4/MUC5AC (the profile resulting from DYNC1LI1 knockdown) benefit more from 5-FU treatment

Several mechanisms may explain this chemosensitivity effect:

• Mucin-mediated pathway: DYNC1LI1 knockdown alters mucin expression profiles, with MUC1 reduction potentially decreasing drug resistance mechanisms

• Cell cycle regulation: As DYNC1LI1 is required for spindle assembly checkpoint progression, altered checkpoint function may affect cell cycle arrest and apoptotic response to DNA-damaging agents

• Intracellular transport alterations: Changes in dynein-mediated transport could impact trafficking of drugs or drug metabolites within cancer cells

These findings suggest DYNC1LI1 could serve as:

• A biomarker for predicting chemotherapy response

• A potential therapeutic target to enhance chemosensitivity

• A component of combination approaches targeting both DYNC1LI1 and mucin expression

What approaches can detect post-translational modifications of DYNC1LI1 and what is their significance?

Post-translational modifications (PTMs) of DYNC1LI1, particularly phosphorylation, appear functionally significant in cellular processes and cancer:

Detection methods:

Significance of DYNC1LI1 phosphorylation:

• The phosphorylated form selectively removes MAD1L1 and MAD1L2 from kinetochores

• This function is critical for proper spindle assembly checkpoint progression

• Increased DYNC1LI1 phosphorylation in prostate cancer suggests disease relevance

Studying DYNC1LI1 PTMs provides insights into cell cycle regulation mechanisms in cancer and may identify potential therapeutic targets affecting cytoskeletal functions crucial for cancer cell division and metastasis.

What methodological approaches can researchers use to study interactions between DYNC1LI1 and other proteins?

Understanding DYNC1LI1's protein interactions requires multiple complementary techniques:

Recommended methodological approaches:

• Co-immunoprecipitation with biotin-conjugated DYNC1LI1 antibody:

  • Use streptavidin-coated beads for efficient capture

  • Identify interacting proteins via mass spectrometry or western blotting

  • Ideal for detecting stable interactions

• Proximity ligation assay:

  • Uses DYNC1LI1 antibody paired with antibodies against suspected partners

  • Generates fluorescent signal when proteins are within 40nm

  • Visualizes interactions within cellular context

• FRET (Förster Resonance Energy Transfer):

  • Requires fluorescently labeled proteins

  • Detects transient interactions and provides spatial information

  • Useful for studying dynamic interactions in living cells

• Cross-linking mass spectrometry:

  • Chemical cross-linking stabilizes protein complexes

  • Provides information about interaction interfaces

  • Particularly valuable for large complexes like dynein

Known DYNC1LI1 interaction partners include components of the dynein complex (DYNC1I1, DYNC1I2, DYNC1H1, DYNLRB1, DYNLT3), cytoskeletal proteins (FLNA, DCTN1), and numerous regulatory proteins . Investigating these interactions can reveal crucial insights into DYNC1LI1's diverse cellular functions and its role in cancer biology.