DYNLT3 Antibody

What is DYNLT3 and what is its cellular function?

DYNLT3 (Dynein Light Chain Tctex-type 3) functions as a non-catalytic accessory component of the cytoplasmic dynein 1 complex. It plays a critical role in linking dynein to cargos and adapter proteins that regulate dynein function . Cytoplasmic dynein 1 acts as a motor for intracellular retrograde motility of vesicles and organelles along microtubules. DYNLT3 is also involved in efficient progression through mitosis by binding to proteins such as BUB3 as part of transport cargo . In specialized cells like melanocytes, DYNLT3 serves as a fundamental regulator of melanosome movement and distribution . The protein has a calculated molecular weight of 13 kDa and consists of 116 amino acids .

Which experimental techniques are most suitable for detecting DYNLT3?

DYNLT3 can be detected using multiple experimental approaches:

For optimal results in Western blot applications, researchers should validate antibody specificity using CRISPR-Cas9 edited cell lysates as negative controls, as demonstrated with ab121209 antibody .

How should DYNLT3 expression be validated in experimental models?

A multi-method approach is recommended to validate DYNLT3 expression:

• Protein level validation: Combine Western blotting with immunohistochemistry and immunofluorescence to confirm protein expression and localization .

• Transcript level validation: Perform qRT-PCR using validated primers such as forward (5′-GCG ATG AGG TTG GCT TCA ATG CTG-3′) and reverse (5′-CAC TGC ACA GGT CAC AAT GTA CTT G-3′) .

• Experimental manipulation: Validate expression changes using knockdown and overexpression models, comparing multiple shRNA constructs or expression vectors .

• Controls: Include appropriate positive controls (U-937 or HeLa cells) and negative controls (CRISPR-Cas9 edited cells lacking DYNLT3) .

How does DYNLT3 expression vary across different cancer types?

DYNLT3 expression exhibits remarkable tissue-specific patterns in cancer, demonstrating opposing roles depending on the cancer type:

This tissue-specific expression pattern suggests that DYNLT3 may function in a context-dependent manner, warranting careful consideration of cellular context in experimental design .

What are the optimal protocols for DYNLT3 knockdown and overexpression?

Based on validated studies, the following methods have proven effective:

For DYNLT3 knockdown:

• shRNA approach: Utilize validated shRNA sequences such as:

  • sh-DYNLT3-1: 5′-CCG GTC TAT ACA GCA TCG TTT AAA TCT CGA GAT TTA AAC GAT GCT GTA TAG ATT TTT G-3′

  • sh-DYNLT3-2: 5′-CCG GTG ATG GAA CCT GTA CCG TAC TCG AGT ACG GTA CAG GTT CCA TCT TTT TG-3′

• siRNA approach: Employ SMARTpool mix of 4 sequences:

  • 5'-CCC ATA ATA TAG TCA AAG A-3'

  • 5'-GGU GGU AAC GAU UAU AAU G-3'

  • 5'-GGG GAA AGC UUA CAA GUA C-3'

  • 5'-CAG AGG AGC CCG UAU GGA U-3'

For DYNLT3 overexpression:

• Lentiviral vector system: Insert human DYNLT3 cDNA into PLVX-IRES-ZsGreen1 vector

• Transfection protocol: Co-transfect with packing plasmids (psPAX2 and pMD2.G) into HEK293T cells using Lipofectamine 2000

• Selection: Use GFP-positive cells with flow cytometry for stable overexpression, or puromycin (2 μg/ml) for selection of knockdown cells

Validation should include Western blotting to confirm successful modulation of protein expression levels .

How does DYNLT3 influence cell migration and invasion in cancer models?

DYNLT3's effect on cell migration and invasion shows cancer type-specific patterns:

In breast cancer (tumor-promoting):

• DYNLT3 overexpression enhances migration and invasion capabilities

• DYNLT3 knockdown suppresses cell growth, migration, and invasion

• Mechanistically linked to EMT markers: Increased N-cadherin and vimentin, decreased E-cadherin

In cervical cancer (tumor-suppressing):

• DYNLT3 overexpression reduces migration and invasion

• DYNLT3 knockdown increases migration and invasion

• Mechanism involves suppression of Wnt signaling pathway and EMT

Experimental approaches to measure these effects:

• Wound healing assays for migration assessment

• Transwell migration and invasion assays

• Western blotting for EMT markers (E-cadherin, N-cadherin, vimentin)

How can researchers reconcile contradictory roles of DYNLT3 in different cancer types?

The contradictory functions of DYNLT3 across cancer types present a significant research challenge. Methodological approaches to address this include:

• Tissue-specific co-factor analysis: Investigate tissue-specific binding partners using co-immunoprecipitation followed by mass spectrometry to identify differential protein interactions in breast versus cervical cancer models .

• Signaling pathway context: Examine DYNLT3's interaction with the Wnt signaling pathway components across cancer types. In cervical cancer, DYNLT3 overexpression decreased expression of Wnt pathway proteins (Dvl2, Dvl3, p-LRP6, Wnt3a, Wnt5a/b, β-catenin) .

• Genetic background consideration: Analyze the effect of DYNLT3 modulation in the context of different genetic backgrounds by using multiple cell lines from each cancer type .

• In vivo validation: Both subcutaneous xenograft tumor models and metastasis models should be employed to validate in vitro findings across cancer types .

• Age-related context: As DYNLT3 is identified as an age-related gene, experiments should control for age-related factors in analyzing its differential effects .

This multi-faceted approach can help elucidate how a single protein exhibits opposing functions in different cancer contexts.

What are the latest techniques for studying DYNLT3's role in cellular transport mechanisms?

Advanced methodologies for investigating DYNLT3's function in cellular transport include:

• Live-cell imaging of melanosome movement: Utilize melanocyte models to track melanosome distribution before and after DYNLT3 knockdown or overexpression. This approach revealed that DYNLT3 knockdown phenocopies the exclusion of pigmented melanosomes from the perinuclear area .

• Quantitative analysis of organelle distribution: Measure the number of perinuclear melanosomes versus total melanosomes to quantify DYNLT3's effect on organelle positioning .

• Co-localization studies: Combine DYNLT3 antibody staining with markers of specific organelles (e.g., Tyrp1 for melanosomes) to analyze spatial relationships .

• CRISPR-Cas9 genome editing: Generate complete DYNLT3 knockout cell lines for functional studies, validating specificity with Western blot analysis .

• Super-resolution microscopy: Apply techniques such as STORM or PALM to visualize DYNLT3's interaction with microtubules and cargo at nanometer resolution.

How can researchers address contradictions in published data about DYNLT3 binding specificities?

Published literature contains contradictory data regarding DYNLT3 binding specificities . To resolve these contradictions, researchers should:

• Employ multiple binding assay methodologies: Combine pull-down assays, yeast two-hybrid screens, and surface plasmon resonance to validate interactions under different experimental conditions.

• Analyze binding kinetics: Determine association and dissociation constants for putative binding partners to quantitatively assess binding affinities.

• Perform competitive binding studies: Test whether reported binding partners compete for the same or different binding sites on DYNLT3.

• Structural biology approaches: Use X-ray crystallography or cryo-EM to determine the precise binding interfaces between DYNLT3 and its partners.

• Functional validation: Confirm the biological relevance of binding interactions through mutational analysis of key binding residues followed by functional assays.

• Replication studies: Independent laboratories should attempt to replicate contradictory findings using standardized protocols to resolve discrepancies.

What experimental design is optimal for studying DYNLT3's role in apoptosis across different cancer models?

Given the opposing effects of DYNLT3 on apoptosis in different cancer types, an optimal experimental design should include:

• Multi-method apoptosis detection:

  • Flow cytometry with Annexin V/PI staining

  • Measurement of cleaved caspase-3 by Western blotting

  • TUNEL assay for DNA fragmentation

• Combination with chemotherapeutic agents: Test whether DYNLT3 modulation affects sensitivity to standard chemotherapeutics (e.g., cisplatin) .

• Time-course experiments: Monitor apoptosis at multiple time points after DYNLT3 modulation to capture both early and late apoptotic events.

• Pathway-specific inhibitors: Use inhibitors of intrinsic and extrinsic apoptotic pathways to determine which mechanism is primarily affected by DYNLT3.

• In vivo validation: Examine apoptotic markers in tumor sections from xenograft models with modulated DYNLT3 expression.

• Multi-cancer type comparison: Directly compare apoptotic responses in breast cancer versus cervical cancer models under identical experimental conditions to validate tissue-specific effects .

This comprehensive approach would help clarify DYNLT3's complex role in regulating programmed cell death across different cancer contexts.

What are the critical quality control steps for DYNLT3 antibody validation?

Rigorous validation of DYNLT3 antibodies should include:

• Specificity testing: Western blot analysis comparing wild-type and DYNLT3 CRISPR-Cas9 edited cell lysates to confirm absence of signal in knockout samples .

• Cross-reactivity assessment: Test antibody against recombinant DYNLT1 and other DYNLT family members to ensure specificity within the protein family.

• Multiple application validation: Confirm performance across different applications (WB, IHC, IF) with appropriate positive controls (U-937, HeLa cells) .

• Lot-to-lot consistency: Test multiple antibody lots to ensure reproducible performance.

• Epitope mapping: Identify the exact epitope recognized by the antibody (e.g., ab121209 targets a recombinant fragment within amino acids 1-100 of human DYNLT3) .

• Sample preparation optimization: Test different fixation methods, antigen retrieval buffers (TE buffer pH 9.0 vs. citrate buffer pH 6.0), and blocking agents .

How should researchers optimize qRT-PCR protocols for DYNLT3 expression analysis?

For accurate quantification of DYNLT3 mRNA expression:

• Validated primer selection: Use experimentally validated primers:

  • Forward: 5′-GCG ATG AGG TTG GCT TCA ATG CTG-3′

  • Reverse: 5′-CAC TGC ACA GGT CAC AAT GTA CTT G-3′

  Alternative primers:

  • Forward: 5′-AACCAGTGGACTGCAAGCAT-3′

  • Reverse: 5′-CCGGTTCTCCCATCTTACGG-3′

• Reference gene selection: Use stable reference genes such as GAPDH (primers: 5'-ACC CAG AAG ACT GTG GAT GG-3' and 5'-CAC ATT GGG GGT AGG AAC AC-3') .

• Protocol optimization:

  • Set 45 cycles with conditions: denaturation at 95°C for 10s, annealing at 55°C for 15s, extension at 70°C for 1 min

  • Prepare cDNA using a one-step RT kit

  • Use 2⁻ΔΔCt method for relative quantification

• Controls and validation:

  • Include no-template controls and no-RT controls

  • Validate with melt curve analysis to confirm amplification specificity

  • Consider using multiple reference genes for normalization