SLC39A6 monoclonal antibody

What is SLC39A6 and what cellular functions does it regulate?

SLC39A6 (also known as ZIP6) is a zinc-influx transporter belonging to the solute carrier family 39. It plays crucial roles in:

• Zinc homeostasis and cellular zinc uptake

• Epithelial-to-mesenchymal transition (EMT) induction

• Cell adhesion regulation through GSK-3β inactivation

• T-cell receptor signaling regulation

• Mitosis initiation through zinc-dependent pathways

• Meiotic progression to metaphase II in oocytes

SLC39A6 functions by mediating cellular zinc uptake, which subsequently affects multiple signaling pathways. When associated with SLC39A10, the heterodimer mediates zinc uptake that triggers EMT. This zinc influx inactivates GSK3B, enabling unphosphorylated SNAI1 to downregulate adherence genes like CDH1, resulting in loss of cell adherence .

What is the tissue distribution pattern of SLC39A6?

SLC39A6 demonstrates variable expression across tissues:

• High expression: Breast, prostate, placenta, kidney, pituitary, and corpus callosum

• Moderate to low expression: Heart and intestine

• Pathological expression: Adenocarcinoma of the lung and cervical carcinoma

• Cancer-specific expression: Frequently upregulated in breast cancer and liver cancer cells

Immunohistochemical analysis reveals that SLC39A6 protein is localized in both cytoplasmic and nuclear compartments of tumor cells, with significant positive correlation between these two expression patterns (R = 0.422, P < 0.0001) .

How should researchers interpret SLC39A6 expression in different cancer types?

The prognostic significance of SLC39A6 varies by cancer type, requiring careful interpretation:

Breast Cancer: High SLC39A6 expression correlates with:

• Better prognosis in ER+ luminal breast cancer (HR 0.678, 95% CI 0.472‒0.972, P = 0.015)

• Features of less aggressive tumors (low grade, low mitotic counts, low nuclear pleomorphism)

• Positive association with other favorable markers (ER, PgR, FOXA1, GATA3)

Liver Cancer: High SLC39A6 expression correlates with:

This dichotomy highlights the tissue-specific nature of SLC39A6 function and emphasizes the importance of contextual interpretation.

What critical validation steps should be performed for SLC39A6 monoclonal antibodies?

Rigorous validation is essential to ensure specificity and performance across applications:

• Western blotting validation:

  • Confirm single band at the predicted molecular weight (85 kDa)

  • Use positive control cell lines (e.g., MCF7) that express SLC39A6

  • Include negative control cell lines (e.g., Jurkat cells) that lack SLC39A6 expression

• Immunohistochemistry validation:

  • Test on full-face tissue sections before using tissue microarrays

  • Verify homogeneous immunohistochemical staining patterns

  • Assess both cytoplasmic and nuclear staining patterns

• Flow cytometry validation:

  • Compare antibody binding to known positive and negative cell lines

  • Quantify binding percentages (e.g., 86% for nanobody vs 80% for commercial antibodies)

• Specificity confirmation:

  • Test cross-reactivity against related proteins

  • Perform ELISA with multiple antigens to confirm selective binding

How should researchers select among different types of anti-SLC39A6 antibodies?

Selection criteria depend on experimental applications and requirements:

Consider antibody format (unconjugated vs. conjugated), storage buffer composition, and whether the antibody is validated as part of a matched pair for multiplex applications .

What are the most critical epitope considerations when selecting SLC39A6 monoclonal antibodies?

The choice of epitope can significantly impact experimental outcomes:

• Extracellular domain antibodies: Essential for flow cytometry of live cells and functional blocking studies

• Intracellular domain antibodies: Suitable for Western blotting and fixed-cell applications

• Conformational epitopes: May be lost in denaturing conditions, affecting Western blot performance

• Linear epitopes: Generally more reliable for denatured protein detection

For therapeutic development or blocking studies, antibodies targeting functional domains involved in zinc transport or protein-protein interactions may be most effective .

What are the optimal protocols for SLC39A6 detection using immunohistochemistry?

Based on published research methods:

• Fixation and embedding:

  • Fix tissue in 4% paraformaldehyde

  • Embed in paraffin using standard protocols

• Antibody selection and dilution:

  • Primary antibody: SLC39A6 rabbit polyclonal antibody (e.g., cat. no. 14236-1-AP)

  • Working dilution: 1:200

• Scoring system:

  • Staining intensity: 0-3 points (0=negative, 1=weak, 2=mild, 3=strong)

  • Percentage of positive cells: 0 (unstained), 1 (1-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%)

  • Final score: Multiply intensity score by percentage score

• Controls:

  • Include positive control tissues known to express SLC39A6

  • Use negative controls by omitting primary antibody

  • Consider both nuclear and cytoplasmic staining patterns

How can researchers optimize Western blot conditions for SLC39A6 detection?

Optimized protocol based on successful detection strategies:

• Sample preparation:

  • Use standard cell lysis buffers containing protease inhibitors

  • Prepare whole cell lysates from SLC39A6-expressing cells (e.g., MCF7)

  • Include non-expressing controls (e.g., Jurkat cells)

• Electrophoresis conditions:

  • Use 8-10% SDS-PAGE gels to resolve the 85 kDa SLC39A6 protein

  • Load 10-20 μg of total protein per lane

• Transfer and antibody incubation:

  • Transfer to nitrocellulose membrane

  • Block with appropriate blocking buffer

  • Incubate with primary anti-SLC39A6 antibody at optimized dilution

  • Use appropriate secondary antibody (anti-rabbit or anti-mouse HRP conjugate)

• Detection considerations:

  • Expect a single specific band at approximately 85 kDa

  • Validate specificity using positive and negative control cell lines

  • Consider both cytoplasmic and nuclear expression patterns

What considerations are important for flow cytometry analysis of SLC39A6?

Key methodological aspects for successful flow cytometry:

• Cell preparation:

  • Use live cells for surface staining (SLC39A6 has extracellular domains)

  • For intracellular staining, use appropriate fixation and permeabilization protocols

• Antibody selection:

  • Choose antibodies validated for flow cytometry applications

  • Consider unconjugated primary antibodies with fluorophore-conjugated secondaries, or directly conjugated antibodies

  • Nanobodies may offer advantages with 86% binding compared to 80% for conventional antibodies

• Controls and validation:

  • Include isotype controls to assess background staining

  • Use known positive (e.g., MCF7) and negative (e.g., Jurkat) cell lines

  • Perform blocking studies to confirm specificity

• Data analysis:

  • Analyze both percentage of positive cells and mean fluorescence intensity

  • Consider dual staining with other markers to identify specific cell populations

How can SLC39A6 antibodies be utilized to study epithelial-to-mesenchymal transition mechanisms?

SLC39A6 plays a critical role in EMT, offering multiple research approaches:

• Co-immunoprecipitation studies:

  • Use SLC39A6 antibodies to pull down protein complexes

  • Identify interaction with SLC39A10 to form functional heterodimers

  • Analyze associations with NCAM1 and focal adhesion complexes

• Pathway analysis:

  • Monitor zinc-dependent inactivation of GSK3B using phospho-specific antibodies

  • Track SNAI1 nuclear translocation following SLC39A6-mediated zinc influx

  • Assess CDH1 (E-cadherin) downregulation as a downstream effect

• Functional blocking studies:

  • Apply SLC39A6-specific nanobodies to block protein function

  • Monitor effects on cell morphology, migration, and invasion

  • Assess zinc influx using fluorescent zinc probes

• Live cell imaging:

  • Use fluorescently-labeled SLC39A6 antibodies for tracking protein redistribution during EMT

  • Correlate with changes in cell morphology and migratory behavior

What approaches exist for functionally blocking SLC39A6 in experimental systems?

Several strategies have been developed:

• Nanobody-based approaches:

  • SLC39A6-specific nanobodies (e.g., C3 nanobody) can detect and block SLC39A6

  • These bind to native cell surface antigen with higher efficiency than commercial antibodies (86% vs 80%)

  • Demonstrated ability to inhibit cell proliferation in MTT assays

• RNAi-based knockdown:

  • siRNA or shRNA targeting SLC39A6 inhibits proliferation, migration, and invasion of liver cancer cells both in vitro and in vivo

• Antibody-drug conjugates:

  • Ladiratuzumab vedotin, an antibody-drug conjugate targeting SLC39A6, disrupts cell microtubules and causes apoptosis

  • Currently in phase II clinical trials

• Small molecule inhibitors:

  • Development is ongoing based on structural understanding of SLC39A6

How can researchers resolve contradictory findings regarding SLC39A6 as a biomarker in different cancer types?

The seemingly contradictory roles of SLC39A6 in different cancers require methodological approaches to resolve:

How are SLC39A6 monoclonal antibodies being developed for therapeutic applications?

Current therapeutic development strategies include:

• Antibody-drug conjugates (ADCs):

  • Ladiratuzumab vedotin targets SLC39A6 to deliver cytotoxic payloads

  • Disrupts cell microtubules causing apoptosis

  • Currently in phase II clinical trials for cancer treatment

• Therapeutic nanobodies:

  • Camelid-derived single-domain antibodies against SLC39A6

  • Demonstrated ability to inhibit cancer cell proliferation

  • Potential advantages in tumor penetration due to small size

• Combined targeting approaches:

  • Dual targeting of SLC39A6 and SLC39A10 to block heterodimer formation

  • Combination with zinc chelators to enhance anti-cancer effects

  • Integration with conventional chemotherapy or hormone therapy

What methodological approaches can help characterize SLC39A6's role in immune regulation?

SLC39A6 participates in T-cell receptor signaling regulation, suggesting several research avenues:

• Immune cell phenotyping:

  • Flow cytometric analysis of SLC39A6 expression in different immune cell populations

  • Correlation with activation markers and functional states

  • Assessment of zinc influx following immune cell activation

• Functional immunology assays:

  • Effects of SLC39A6 blocking on T-cell proliferation and cytokine production

  • Impact on antigen presentation and immune synapse formation

  • Role in lymphocyte migration and tissue infiltration

• In vivo immune response models:

  • Effects of SLC39A6 manipulation on anti-tumor immunity

  • Influence on autoimmune disease progression

  • Impact on vaccine responses

Understanding these pathways may reveal new immunotherapeutic approaches targeting zinc homeostasis in immune cells.