SLC16A4 Antibody

What is SLC16A4 and what is its biological significance in cellular metabolism?

SLC16A4 (Solute Carrier Family 16 Member 4), also known as MCT5 (Monocarboxylate Transporter 5) or MCT4, is a multi-pass membrane protein that plays a crucial role in cellular metabolism. It functions as a proton-linked monocarboxylate transporter that catalyzes the rapid transport of various monocarboxylates across the plasma membrane . These substrates include:

• Lactate

• Pyruvate

• Branched-chain oxo acids derived from leucine, valine, and isoleucine

• Ketone bodies including acetoacetate, beta-hydroxybutyrate, and acetate

SLC16A4 possesses 12 transmembrane alpha helices and is predominantly localized in the cell membrane . The protein is critical for maintaining cellular energy homeostasis, particularly in environments with fluctuating metabolic demands or hypoxic conditions. Recent research indicates that SLC16A4 does not operate in isolation but forms part of a transport complex, often partnering with ancillary proteins such as basigin (CD147) for optimal function .

What are the standard applications for SLC16A4 antibodies in research?

SLC16A4 antibodies serve as vital tools for investigating this transporter's expression, localization, and function across various experimental contexts. Based on validation data from multiple sources, these antibodies can be reliably used in the following applications:

These applications enable researchers to investigate SLC16A4's role in various biological contexts, from basic protein expression analysis to complex studies of metabolic pathways in cancer and other diseases .

What species reactivity is available for commercial SLC16A4 antibodies?

Most commercially available SLC16A4 antibodies demonstrate reactivity with human samples, though many cross-react with other species due to sequence conservation. The following table summarizes the validated and predicted reactivity patterns based on available antibody products:

When selecting an SLC16A4 antibody for cross-species applications, researchers should carefully review validation data for their specific species of interest, as theoretical reactivity predictions may not always translate to experimental success .

How should researchers optimize Western blot protocols for SLC16A4 detection?

Optimizing Western blot protocols for SLC16A4 detection requires attention to several critical parameters:

Sample Preparation and Loading:

• Use appropriate positive control samples such as rat testis tissue, JURKAT cells, HeLa cells, or MCF-7 cells

• Standard protein extraction buffers with protease inhibitors are generally suitable

• Load 20-50 μg of total protein per lane for cell/tissue lysates

Electrophoresis and Transfer Parameters:

• SLC16A4 has a calculated molecular weight of 54 kDa, though observed bands may appear at 54-60 kDa depending on post-translational modifications

• Use 8-12% polyacrylamide gels for optimal resolution

• Standard transfer conditions are generally effective

Antibody Incubation and Detection:

• Primary antibody dilutions typically range from 1:500 to 1:2000 based on specific product recommendations

• Overnight incubation at 4°C often yields optimal results

• Use appropriate species-specific HRP-conjugated secondary antibodies

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

Troubleshooting Common Issues:

• Multiple bands may indicate splice variants, post-translational modifications, or degradation products

• High background may require increased blocking time or higher BSA/milk concentrations

• Weak signal may necessitate longer exposure times or increased antibody concentrations

When validating a new SLC16A4 antibody, comparing results with multiple antibodies targeting different epitopes can help confirm specificity and rule out non-specific binding .

What are important considerations for immunohistochemical detection of SLC16A4?

Immunohistochemical analysis of SLC16A4 requires careful consideration of fixation, antigen retrieval, and staining optimization:

Tissue Processing and Antigen Retrieval:

• Formalin fixation and paraffin embedding is standard for most applications

• Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective

• Optimal antigen retrieval time may vary based on fixation duration

Staining Pattern Interpretation:

• In normal pancreatic tissue, SLC16A4 typically shows negative staining in the nucleus, cytoplasm, and cell membrane

• In pancreatic cancer samples, staining patterns are distinguishable with strong staining positive on the cell membrane

• Weak patterns in cancer may localize to the nucleus, while moderate and strong patterns typically localize to the cell membrane

Staining Intensity Classification:
Based on Human Protein Atlas data for pancreatic tissue samples:

• Normal tissue: Predominantly weak staining (all cases)

• Cancer tissue: Variable intensity including weak, moderate, and strong staining

  • Weak: ~17% of cases

  • Moderate: ~58% of cases

  • Strong: ~25% of cases

These distinct staining patterns can help differentiate normal from cancerous tissue and potentially correlate with clinical outcomes in research settings .

How is SLC16A4 expression altered in pancreatic cancer, and what are the implications for using SLC16A4 antibodies in cancer studies?

SLC16A4 shows significant expression alterations in pancreatic cancer compared to normal pancreatic tissue, making it a valuable target for cancer research applications. Comprehensive analysis of SLC16A family members in pancreatic cancer reveals the following:

Expression Profile in Pancreatic Cancer:

• SLC16A4 shows significantly increased expression in pancreatic adenocarcinoma (PAAD) compared to normal pancreatic tissue

• Among the SLC16A family, SLC16A1, SLC16A3, and SLC16A4 demonstrate the most notable alterations in pancreatic cancer

• RNA-Seq data from 179 pancreatic cancer vs. 171 normal pancreas samples confirms statistically significant upregulation of SLC16A4

Methodological Approaches for Expression Analysis:

• Immunohistochemistry: SLC16A4 antibodies can be used to distinguish between normal and cancerous pancreatic tissues based on staining intensity and subcellular localization patterns

• Western blotting: Can quantify relative expression levels across pancreatic cancer cell lines compared to normal pancreatic tissue

• RNA interference studies: Knockdown experiments have demonstrated that SLC16A4 alterations can influence the expression of other SLC16A family members, suggesting complex regulatory networks

Research Implications:
These expression differences suggest that SLC16A4 antibodies can be valuable tools for:

• Identifying metabolic alterations in pancreatic cancer tissues

• Exploring the role of monocarboxylate transport in tumor microenvironment development

• Investigating potential therapeutic targets related to cancer metabolism

• Developing potential diagnostic or prognostic markers for pancreatic cancer

What is the prognostic value of SLC16A4 in cancer studies, and how can antibodies help evaluate this?

Analysis of The Cancer Genome Atlas (TCGA) data indicates that SLC16A4 may have prognostic significance in certain cancer types. While SLC16A4 itself hasn't shown as strong a prognostic correlation as some other family members, several SLC16A proteins have demonstrated significant associations with patient outcomes.

Prognostic Associations in Pancreatic Cancer:

Methodological Approaches for Prognostic Studies:
Researchers can utilize SLC16A4 antibodies in prognostic investigations through:

• Tissue Microarray Analysis:

  • Staining intensity classification (weak, moderate, strong)

  • Subcellular localization assessment (membrane vs. cytoplasmic vs. nuclear)

  • Correlation with clinical outcomes and other prognostic markers

• Multi-marker Panels:

  • Combined with other SLC16A family antibodies for comprehensive metabolic profiling

  • Integration with established prognostic markers

• Quantitative Analysis:

  • Digital image analysis of immunohistochemistry

  • H-score calculation based on staining intensity and percentage of positive cells

One validated approach uses a 9-gene model that includes SLC16A4 along with VHL, PTGER4, HK1, DLL4, CXCL12, CXCR4, PTGER3, and CA9 to classify newly diagnosed head and neck squamous cell carcinoma .

How can SLC16A4 antibodies be used to investigate protein-protein interactions and molecular complexes?

SLC16A4 functions within multiprotein complexes, and antibodies provide powerful tools for investigating these interactions. Research has shown that SLC16A4 specifically interacts with β1 integrin, potentially regulating cell migration through modulation of focal adhesions .

Methodological Approaches:

• Co-immunoprecipitation (Co-IP):

  • Use SLC16A4 antibodies to pull down protein complexes

  • Western blot analysis with antibodies against suspected interacting partners

  • Reverse Co-IP with partner antibodies to confirm interactions

• Proximity Ligation Assay (PLA):

  • Combines antibody specificity with PCR amplification

  • Enables visualization of protein interactions in situ

  • Requires antibodies from two different species targeting SLC16A4 and potential binding partners

• Immunofluorescence Co-localization:

  • Double immunofluorescence staining using SLC16A4 antibodies alongside antibodies for suspected interacting proteins

  • Confocal microscopy analysis with co-localization quantification

  • Particularly useful for examining membrane localization patterns

• FRET/BRET Studies:

  • More sophisticated approaches requiring fluorescent/bioluminescent protein tags

  • Can be combined with antibody-based techniques for validation

Research Applications:

• Investigation of SLC16A4's role in focal adhesion dynamics at the leading edge of migrating cells

• Examination of transport complex formation with ancillary proteins

• Study of signaling pathways influenced by SLC16A4-protein interactions in metabolic regulation

What are the best experimental approaches for studying SLC16A4's role in cellular metabolism using antibody-based techniques?

SLC16A4 plays a crucial role in monocarboxylate transport and cellular metabolism, particularly in cancer contexts where metabolic reprogramming is a hallmark feature. Antibody-based approaches offer several avenues for investigating these metabolic functions.

Experimental Strategies:

• Metabolic Flux Analysis with SLC16A4 Knockdown/Overexpression:

  • Use SLC16A4 antibodies to confirm knockdown/overexpression efficiency

  • Measure lactate production/consumption rates

  • Trace metabolic pathways using isotope-labeled substrates

  • Quantify changes in mitochondrial vs. glycolytic metabolism

• Correlation of SLC16A4 Expression with Metabolic Markers:

  • Multiplex immunohistochemistry/immunofluorescence combining SLC16A4 antibodies with:

    • Glycolytic enzymes (HK2, PKM2, LDHA)

    • Hypoxia markers (HIF-1α, CAIX)

    • Mitochondrial markers (TOMM20, COX4)

• Live-Cell Imaging of Metabolite Transport:

  • Combine fluorescent metabolite analogs with SLC16A4 immunostaining

  • Real-time visualization of transport activity in relation to protein localization

• Cell-Based Metabolic Assays Following SLC16A4 Modulation:

  • Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Correlate with SLC16A4 expression levels determined by immunoblotting

  • Assess metabolic flexibility under different substrate conditions

Research Implications:
These approaches can reveal:

• How SLC16A4 influences the Warburg effect in cancer cells

• Metabolic adaptations following SLC16A4 inhibition

• The role of SLC16A4 in maintaining pH homeostasis under hypoxic conditions

• Potential metabolic vulnerabilities that could be therapeutically targeted

How is SLC16A4 involved in cell migration and what techniques using antibodies can elucidate this function?

Recent research has uncovered an unexpected role for SLC16A4 in regulating cell migration that extends beyond its canonical metabolite transport function. Studies indicate that SLC16A4 interacts with β1 integrin, potentially modulating focal adhesion dynamics and directed cell migration .

Mechanistic Insights:

• SLC16A4 expression at the leading edge of migrating cells may relieve intracellular acid load

• This allows glycolysis to continue uninterrupted, providing energy for migration

• Lactate efflux via SLC16A4 could stabilize integrin-mediated attachment

• These mechanisms may be particularly relevant in pathological contexts such as proliferative vitreoretinopathy (PVR) and metastatic cancer

Antibody-Based Investigative Approaches:

• Time-Lapse Immunofluorescence of Migrating Cells:

  • Track SLC16A4 localization during different phases of cell migration

  • Correlate with focal adhesion markers and migration parameters

• Wound Healing Assays with SLC16A4 Immunostaining:

  • Assess localization patterns at the wound edge

  • Quantify migration rates in relation to expression levels

  • Compare effects of SLC16A4 knockdown or inhibition

• 3D Migration and Invasion Assays:

  • Examine SLC16A4 expression in invadopodia

  • Correlate with matrix degradation capability

  • Analyze influence on 3D migration patterns

• Integrin-SLC16A4 Co-localization Studies:

  • Use proximity ligation assays to visualize interactions

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics

  • Investigate effects of disrupting this interaction on migration

These approaches can provide valuable insights into how SLC16A4 contributes to the migratory phenotype of cancer cells, potentially revealing new therapeutic strategies targeting metastasis.

What are the latest findings on SLC16A4's role in chromatin modification and DNA damage repair?

Emerging research has begun to uncover unexpected nuclear functions of SLC16A4 that extend beyond its canonical role as a membrane transporter. Recent findings suggest potential involvement in chromatin modification and DNA damage repair mechanisms .

Key Research Findings:

• SLC16A4 genetic aberrations may influence carcinogenesis through effects on chromatin organization

• Integrative analysis with in vitro functional data and animal models has provided supportive evidence for these non-canonical roles

• The weak nuclear staining pattern observed in some cancer cells may be functionally significant rather than artifactual

Investigative Approaches Using Antibodies:

• Chromatin Immunoprecipitation (ChIP):

  • Use SLC16A4 antibodies to identify potential chromatin binding sites

  • Couple with sequencing (ChIP-seq) for genome-wide binding analysis

  • Validate findings with site-specific PCR

• Immunofluorescence Co-localization with DNA Damage Markers:

  • Co-stain for SLC16A4 and DNA damage response proteins (γ-H2AX, 53BP1, RAD51)

  • Assess nuclear localization following DNA damage induction

  • Quantify recruitment to damage sites

• Proximity-Based Proteomic Approaches:

  • BioID or APEX2-based proximity labeling with SLC16A4

  • Mass spectrometry identification of nuclear interaction partners

  • Validation with co-immunoprecipitation using SLC16A4 antibodies

• Live-Cell Imaging of Nuclear SLC16A4 Dynamics:

  • Track SLC16A4 localization during cell cycle progression

  • Monitor response to DNA damaging agents

  • Assess co-localization with chromatin markers

These emerging research directions could significantly expand our understanding of SLC16A4's cellular functions beyond metabolite transport, potentially revealing new therapeutic opportunities in cancer and other diseases.