tmem47 Antibody

What is TMEM47 and what are its key cellular functions?

TMEM47 (Transmembrane Protein 47) is a member of the PMP22/EMP/Claudin protein family. The canonical human TMEM47 protein consists of 181 amino acid residues with a molecular mass of approximately 20 kDa . It primarily localizes to the endoplasmic reticulum (ER) and plasma membrane .

TMEM47 has several established functions:

• Regulates cell junction organization in epithelial cells

• May play a crucial role in the transition from adherens junction to tight junction assembly

• Potentially regulates F-actin polymerization required for tight junctional localization dynamics

• Affects the junctional localization of PARD6B

• During podocyte differentiation, may negatively regulate activity of FYN and subsequently the abundance of nephrin

Notably, TMEM47 is the vertebrate orthologue of C. elegans VAB-9, a tetraspan adherens junction protein that regulates cell morphology and adhesion .

What is the tissue distribution pattern of TMEM47 expression?

TMEM47 exhibits tissue-specific expression patterns. According to current research, TMEM47 is primarily expressed in:

• Adult brain

• Fetal brain

• Cerebellum

• Heart

• Lung

• Prostate

• Thyroid

Additionally, TMEM47 expression has been documented during kidney development and podocyte differentiation .

How is TMEM47 expression regulated during viral infections?

Research using zebrafish models has demonstrated that tmem47 expression is rapidly upregulated during both RNA and DNA virus infections. In zebrafish embryonic fibroblast (ZF4) cells, tmem47 expression increased after infection with SVCV (Spring Viremia of Carp Virus) or CyHV-2 (Cyprinid herpesvirus 2), reaching peak levels at 48 hours post-infection. This temporal pattern coincided with the upregulation of key antiviral genes including ifnφ1 and irf7 .

Similar induction patterns were observed in zebrafish liver (ZFL) cells infected with these viruses, suggesting TMEM47 plays a role in antiviral responses .

What experimental applications are TMEM47 antibodies most commonly used for?

Based on current research protocols, TMEM47 antibodies are primarily used in the following applications:

For optimal results in zebrafish research, specific anti-tmem47 antibodies suitable for Western blot and ELISA applications are available .

What are the preferred methods for validating TMEM47 expression changes in experimental settings?

For robust validation of TMEM47 expression changes, researchers typically employ complementary techniques:

• mRNA quantification by qRT-PCR:

  • RNA extraction using TRIZOL Reagent

  • cDNA synthesis using PrimeScript™ RT reagent Kit with gDNA Eraser Kit

  • qRT-PCR using SYBR green dye method

  • Relative quantification using 2^-ΔΔCt method with appropriate housekeeping genes (18S rRNA for tissue samples, β-actin for cell lines)

• Protein validation by Western blotting:

  • Protein extraction using Cell Lysis Buffer

  • Quantification with Bio-Rad Protein Assay Dye Reagent

  • SDS-PAGE using 12% bis-acrylamide gels

  • Transfer to PVDF membranes

  • Blocking with 5% non-fat milk

  • Primary antibody incubation (typically 1:1,000 dilution) at 4°C overnight

  • Detection using ECL Prime Western Blotting Detection kit

How can I effectively overexpress or knockdown TMEM47 for functional studies?

Based on published research methodologies:

For TMEM47 overexpression:

• Lentiviral open reading frame (ORF) clones are effective for stable expression

• The sequence can be cloned into vectors like pLOC

• Viral particles can be produced by transfection into 293T cells

• Selection with appropriate antibiotics (e.g., blasticidin) following transduction

• Confirmation of expression by qRT-PCR and Western blotting

For TMEM47 knockdown:

• siRNA or shRNA approaches are both effective

• For transient knockdown: siRNAs targeting TMEM47 can be transfected using liposome reagents

• For stable knockdown: lentiviral shRNA particles (such as Mission® shRNA) can be used

• Control transfections should include non-target control siRNA/shRNA

• Validation of knockdown efficiency through qRT-PCR and Western blotting

How does TMEM47 contribute to chemoresistance in hepatocellular carcinoma (HCC)?

TMEM47 has been identified as a significant contributor to chemoresistance in HCC. Key research findings include:

• TMEM47 expression is significantly upregulated in HCC cell lines with acquired chemoresistance

• Patients not responding to cisplatin-based transarterial chemoembolization (TACE) treatment show significantly higher TMEM47 expression compared to responders

• TMEM47 mRNA expression levels positively correlate with the degree of cisplatin resistance in HCC cells

• Overexpression of TMEM47 in HCC cells significantly promotes cisplatin resistance

• Targeted inhibition of TMEM47 can significantly reduce cisplatin resistance via:

  • Enhancing caspase-mediated apoptosis

  • Suppressing cisplatin-induced activation of genes involved in drug efflux and metabolism

• TMEM47 expression significantly correlates with multi-drug resistance-associated protein 1 in patients with HCC receiving TACE treatment

These findings suggest TMEM47 could serve as both a biomarker for predicting chemotherapy response and a potential therapeutic target for overcoming HCC chemoresistance.

What is the role of TMEM47 in tamoxifen resistance in breast cancer?

Research has demonstrated that TMEM47 significantly contributes to tamoxifen (TAM) resistance in breast cancer:

• TMEM47 mRNA is dramatically upregulated (Log2FC = 7.12) in tamoxifen-resistant MCF-7 cells (TAMR/MCF-7)

• Protein levels are similarly increased in resistant cells

• Stable overexpression of TMEM47 in MCF-7 cells (TMEM47-OE/MCF-7) confers resistance to tamoxifen

• The IC50 values for 4-hydroxy-tamoxifen (4OH-TAM) are:

  Cell Line	IC50 (μg/mL)	Resistance Index
  MCF-7	1.58 ± 0.19	/
  TAMR/MCF-7	2.74 ± 0.24	1.74
  Lenti-control/MCF-7	1.36 ± 0.27	/
  TMEM47-OE/MCF-7	3.12 ± 0.32	2.30

• TMEM47 overexpression suppresses apoptosis in breast cancer cells exposed to tamoxifen

• Knockdown of TMEM47 in resistant cells significantly enhances sensitivity to tamoxifen:

  • Cell viability decreased to 52% and 39% after 24 and 48 hours of tamoxifen treatment

  • Apoptosis rates increased significantly

These findings suggest TMEM47 could be a potential biomarker and therapeutic target for tamoxifen resistance in breast cancer.

What are the known associations of TMEM47 with other diseases?

According to GeneCards, TMEM47 has been associated with:

• Dandy-Walker Syndrome

• Attention Deficit-Hyperactivity Disorder

While the specific mechanistic roles in these conditions require further investigation, TMEM47's involvement in neurological conditions aligns with its high expression in brain tissues.

How does TMEM47 regulate interferon production during viral infections?

Research in zebrafish has revealed that TMEM47 acts as a negative regulator of interferon (IFN) production during both RNA and DNA viral infections. The mechanism involves:

• TMEM47 physically interacts with MAVS (Mitochondrial Antiviral Signaling protein) and STING (Stimulator of Interferon Genes)

• These interactions promote the degradation of MAVS and STING through an autophagy-lysosome-dependent pathway

• The autophagy factor ATG5 (Autophagy-related Gene 5) is essential for this degradation process

• Overexpression of TMEM47 significantly blocks virus-mediated IFN induction

• Knockdown of tmem47 promotes ifn transcription during viral infection

• At the cellular level, MAVS- and STING-mediated antiviral capacities are significantly suppressed by TMEM47

This mechanism demonstrates that TMEM47 serves as a feedback regulator to maintain homeostasis of cellular IFN responses during viral infections.

What is known about TMEM47's role in cell junction dynamics?

TMEM47 plays a sophisticated role in epithelial cell junction dynamics:

• Unlike claudins, TMEM47 does not generate tight junction strands when expressed in L fibroblasts

• TMEM47 membrane localization requires E-cadherin expression

• Temporally, TMEM47 localizes at cell junctions first with E-cadherin before ZO-1 colocalization

• In polarized epithelia, TMEM47 colocalizes with adherens junction proteins

• By immunoprecipitation, TMEM47 associates with both:

  • Classical adherens junction proteins

  • Tight junction proteins Par6B and aPKCλ

Functional studies have shown that:

• Overexpression of TMEM47 in MDCK cells:

  • Decreases apical surface area

  • Increases activated myosin light chain at cell-cell contacts

  • Disrupts cell polarity and morphology

  • Delays cell junction reassembly following calcium switch

  • Selectively interferes with tight junction assembly

• Reduced TMEM47 expression results in opposite phenotypes

These findings suggest TMEM47 regulates the localization of a subset of tight junction proteins, associated actomyosin structures, cell morphology, and participates in developmental transitions from adherens to tight junctions.

What experimental approaches have been used to improve TMEM47 antibody thermostability?

While not directly addressing TMEM47 antibodies specifically, research on improving antibody thermostability has employed:

• Consensus sequence-based methods:

  • Typically shows a success rate of about 50%

  • Maximum melting temperature increments range from 10 to 32°C

• Combined structural-consensus approaches:

  • Adding a 3D structural layer to the consensus sequence method

  • Analyzing conserved close-by residue pairs in antibody 3D structures

  • Developing scoring systems for favorable interactions:

    • Charge score = charge score(residue 1) * charge score(residue 2)

    • Hydrophobicity score = -1 * hydrophobicity score(residue 1) * hydrophobicity score(residue 2)

    • Aromaticity score = -1 * aromaticity score(residue 1) * aromaticity score(residue 2)

    • Total score = charge score + hydrophobicity score + aromaticity score

  • This structural method significantly decreases false positives from consensus sequence method prediction

These approaches could potentially be applied to develop more thermostable TMEM47 antibodies for research applications.

How conserved is TMEM47 across different species?

TMEM47 demonstrates remarkable evolutionary conservation:

• Human TMEM47 shares 82% identity with zebrafish TMEM47

• TMEM47 gene orthologs have been reported in:

  • Mouse

  • Rat

  • Bovine

  • Frog

  • Zebrafish

  • Chimpanzee

  • Chicken

  • Japanese flounder (Paralichthys olivaceus)

The high degree of conservation suggests critical functional roles that have been maintained throughout vertebrate evolution.

Do TMEM47 functions differ between species, and how does this impact antibody selection?

Despite high conservation, some functional differences exist:

• Zebrafish TMEM47 has been demonstrated to negatively regulate interferon production during viral infections by targeting MAVS and STING for degradation

• Vertebrate TMEM47 is the orthologue of C. elegans VAB-9, and expression of TMEM47 in C. elegans functionally rescues vab-9 mutations

• In mammals, TMEM47 regulates epithelial cell junction maturation and morphogenesis

For antibody selection, researchers should consider:

• Species-specific antibodies when studying TMEM47 in non-human models

• The potential for cross-reactivity due to high sequence conservation

• The specific applications (Western blot, ELISA, immunoprecipitation) needed

• Whether functional studies will involve TMEM47 from different species

What are the most effective methods for studying TMEM47 function across different experimental models?

Based on published research, effective methods include:

For in vitro studies:

• Cell line models with appropriate TMEM47 expression

• CRISPR/Cas9 knockout models

• Overexpression and knockdown approaches via lentiviral vectors

• Functional assays specific to the pathway being studied (e.g., drug resistance, interferon production)

For analyzing protein-protein interactions:

• Co-immunoprecipitation with appropriate antibodies

• Confocal microscopy for colocalization studies

• Proximity ligation assays

For in vivo studies:

• Zebrafish models have proven effective for studying TMEM47's role in viral infections

• Mouse models for studying developmental roles and mammalian-specific functions

For cross-species functional studies:

• Expression of TMEM47 from one species in another (such as the successful rescue of C. elegans vab-9 mutations with vertebrate TMEM47)

• Comparative analyses of TMEM47 function in different cell lines derived from various species