DYNLL1 Antibody

What is DYNLL1 and what are its basic characteristics?

DYNLL1 is a 10.4 kDa protein (89 amino acids in humans) belonging to the dynein light chain protein family. It functions as a non-catalytic accessory component of the cytoplasmic dynein 1 complex involved in linking dynein to cargoes and adapter proteins. DYNLL1 is ubiquitously expressed and localizes to the nucleus, mitochondria, and cytoplasm. Several synonyms exist in the literature, including DLC8, DNCL1, DNCLC1, LC8, LC8a, PIN, hdlc1, and DLC1, with orthologs reported in mouse, rat, bovine, frog, and chimpanzee species .

What should researchers consider when selecting DYNLL1 antibodies?

When selecting DYNLL1 antibodies, researchers should consider:

• Target epitope: Some antibodies target specific regions (e.g., N-terminal half)

• Antibody format: Monoclonal antibodies offer greater specificity and reproducibility compared to polyclonal options

• Validated applications: Different antibodies are validated for specific techniques, including Western blot, immunohistochemistry, immunocytochemistry, and ELISA

• Species reactivity: Confirm cross-reactivity with your experimental model (human, mouse, rat, etc.)

• Clone specificity: For instance, ZooMAb rabbit recombinant monoclonal antibodies offer enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals

What are the most common applications for DYNLL1 antibodies?

DYNLL1 antibodies are employed across several methodologies:

• Western Blot: Most widely used application for detecting DYNLL1 protein expression

• Immunohistochemistry (Paraffin): Used to visualize DYNLL1 in tissue sections (e.g., human liver tissue)

• Immunocytochemistry/Immunofluorescence: Detecting subcellular localization in cell lines (e.g., MCF-7 cells)

• ELISA: For quantitative measurements of DYNLL1 levels

• Immunoprecipitation: Isolating DYNLL1 protein complexes for interaction studies

How does DYNLL1 regulate NF-κB signaling pathways?

DYNLL1's role in NF-κB signaling has undergone significant revision. While early overexpression studies suggested DYNLL1 directly inhibits IκBα phosphorylation and degradation, recent physiological studies demonstrate DYNLL1 is actually required for signal-specific activation of the NF-κB pathway upstream of IκBα. Specifically:

• Under physiological conditions, DYNLL1 promotes signal-specific activation of IKK

• DYNLL1 is essential for TLR4- and IL-1-mediated activation of the NF-κB pathway in mouse B cells and fibroblasts

• DYNLL1 acts upstream of IκBα in the signaling cascade

This apparent contradiction highlights the importance of studying DYNLL1 at physiological expression levels rather than overexpression systems.

What is the relationship between DYNLL1 and B-cell development/antibody responses?

DYNLL1 plays critical roles in B-cell biology:

• Deletion of Dynll1 during early B-cell development leads to approximately 10-fold reduced numbers of mature follicular B cells (B2 cells) and >100-fold reduction of innate-like B-1a cells

• DYNLL1 is essential for TLR4-mediated B-cell activation and antibody responses

• DYNLL1 contributes to immunoglobulin gene class switch recombination in mature B cells by modulating oligomerization of the DNA damage response protein 53BP1

• The B2 (but not B-1a) cell developmental defects can be suppressed by simultaneous deletion of the DYNLL1 target BIM

Researchers demonstrated the in vivo significance of DYNLL1 through impaired T-cell-independent type 1 (TI-1) antibody responses in DYNLL1-deficient mice.

How can researchers experimentally assess DYNLL1's function in immune responses?

Methodological approaches include:

• Conditional knockout models: Using systems like Cd23-Cre or Mb1-Cre to delete Dynll1 at different B-cell developmental stages

• Immunization protocols: Assessing TI-1 responses through NP-LPS immunization and measuring antigen-specific antibody titers

• Biochemical analysis: Monitoring IκBα protein levels and degradation kinetics in LPS-treated B cells via immunoblotting

• Ex vivo assays: Analyzing LPS-induced B-cell proliferation and survival using flow cytometry and metabolic assays

What experimental controls are essential when using DYNLL1 antibodies?

Critical controls include:

• Positive control: Lysates from cells known to express DYNLL1 (e.g., MCF-7 cells)

• Negative control: Lysates from DYNLL1-knockout cells or tissues

• Antibody validation: Confirmation using recombinant DYNLL1 protein (e.g., His/ABP-tagged recombinant fragment)

• Blocking peptide: Competition assay with the immunizing peptide

• Loading control: Using housekeeping proteins (β-actin, GAPDH) to normalize expression levels

• Isotype control: For immunohistochemistry and immunofluorescence applications

How should researchers optimize Western blot protocols for DYNLL1 detection?

For optimal Western blot results:

• Sample preparation: Use RIPA buffer with protease inhibitors to extract total protein

• Protein loading: Load 20-30 μg of total protein per lane

• Gel selection: Use 15-20% polyacrylamide gels to resolve small proteins (~10 kDa)

• Transfer conditions: Semi-dry transfer with PVDF membrane (0.2 μm pore size) for 30 minutes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour

• Primary antibody: Dilute 1:1000 and incubate overnight at 4°C

• Detection system: Enhanced chemiluminescence with extended exposure time for low-abundance samples

What approaches can be used to study DYNLL1 protein-protein interactions?

Researchers can employ multiple complementary techniques:

• Co-immunoprecipitation: Using DYNLL1 antibodies to pull down interacting partners

• Yeast two-hybrid screening: To identify novel interaction partners

• GST-pulldown assays: With recombinant DYNLL1 as bait

• Proximity ligation assay: For visualizing interactions in situ

• Bioinformatic analysis: Identifying proteins containing the (K/R)XTQT consensus binding motif

• Molecular docking: Computational prediction of interactions using tools like PatchDock and FireDock

What is known about DYNLL1's role in cancer and chemoresistance?

Research has revealed important connections between DYNLL1 and cancer:

• Deletion of DYNLL1 increases chemoresistance in BRCA1-mutant high-grade serous ovarian carcinoma cells

• DYNLL1 expression is elevated in ovarian cancer histotypes compared to normal ovarian cells

• Expression patterns differ by cell type: decreased in epithelial-type OC cells but increased in stromal-type OC cells

• DYNLL1 expression patterns are associated with chemosensitivity profiles in ovarian cancer

• These findings suggest DYNLL1 could be a potential biomarker or therapeutic target for overcoming chemoresistance

How might DYNLL1 be involved in pathogen-host interactions?

Emerging evidence suggests DYNLL1 may play roles in bacterial and viral infections:

• DYNLL1 interacts with viral proteins via a short linear consensus motif sequence (K/R)XTQT

• Computational analyses predict similar interactions with bacterial proteins, including:

  • Enterochelin esterase from Salmonella typhimurium

  • Protective antigen from Bacillus anthracis

• These interactions may facilitate pathogen intracellular transport and contribute to infection

• Molecular docking analyses support the potential for these protein-protein interactions

• This represents a novel area for therapeutic intervention targeting pathogen-host interactions

How can researchers reconcile contradictory findings regarding DYNLL1's role in NF-κB signaling?

The apparent contradiction between DYNLL1 as an inhibitor versus activator of NF-κB requires careful experimental design:

• Expression level considerations: Early studies used overexpression systems, while recent work examines physiological expression levels

• Cell type specificity: Different cellular contexts may influence DYNLL1 function

• Stimulus specificity: DYNLL1 appears to regulate signal-specific NF-κB activation (particularly TLR4 and IL-1 pathways)

• Time-course analysis: Monitor NF-κB activation kinetics rather than single timepoints

• Genetic approaches: Use conditional knockout models rather than transient knockdown/overexpression

• Direct biochemical assays: Examine IκBα phosphorylation, degradation, and IKK activation in physiologically relevant contexts

What unanswered questions remain regarding DYNLL1 function in immune responses?

Several important knowledge gaps exist:

• The molecular mechanism by which DYNLL1 promotes IKK activation remains undefined

• Whether DYNLL1 regulates other PRR-dependent signaling pathways beyond TLR4/IL-1

• The full complement of DYNLL1-interacting proteins in immune cells

• How DYNLL1-dependent signaling intersects with other B-cell activation pathways

• The potential therapeutic applications of targeting DYNLL1 in inflammatory diseases

What technical innovations might advance DYNLL1 research?

Emerging technologies that could enhance DYNLL1 studies include:

• CRISPR-Cas9 gene editing: Creating precise mutations in DYNLL1 binding domains

• Single-cell analysis: Examining DYNLL1 function in heterogeneous immune cell populations

• Proximity-dependent labeling: Capturing transient DYNLL1 interactions in living cells

• Cryo-electron microscopy: Resolving DYNLL1-containing complexes at high resolution

• Phospho-proteomics: Identifying signaling cascades regulated by DYNLL1

• In vivo imaging: Tracking DYNLL1 dynamics during immune responses