SMTNL1 Antibody

What is SMTNL1 and what are its primary biological functions?

SMTNL1 (Smoothelin-like protein 1) is a transcriptional cofactor that plays significant roles in multiple biological processes. It functions primarily as a regulator of skeletal muscle phenotype, mediating the transition from oxidative to glycolytic fiber types particularly during pregnancy. SMTNL1 interacts with the progesterone receptor to alter the expression of contractile and metabolic proteins, contributing to physiological adaptations in skeletal muscle . Additionally, SMTNL1 has been identified as an important factor in glucose homeostasis and insulin signaling. Research has shown that SMTNL1 can attenuate insulin resistance by regulating IRS-1 serine phosphorylation and enhancing GLUT4-mediated glucose uptake . In endometrial tissue, SMTNL1 promotes epithelial cell differentiation in a progesterone-dependent manner and inhibits cell migration under hyperglycemic conditions, suggesting potential roles in female reproductive physiology and gestational diabetes .

What experimental models are commonly used to study SMTNL1 function?

Based on the research literature, several experimental models have been established to investigate SMTNL1 function:

• Transgenic mouse models: SMTNL1 knockout (smtnl1 −/−) mice serve as valuable in vivo models for studying the physiological role of SMTNL1. These models allow researchers to examine the effects of SMTNL1 deletion on metabolism, pregnancy outcomes, and glucose tolerance .

• Cell culture systems:

  • C2C12 rodent skeletal muscle cells are used to study SMTNL1's role in insulin signaling and glucose metabolism

  • Ishikawa endometrial epithelial cells are employed to investigate SMTNL1's effects on cell differentiation, particularly in response to progesterone and in gestational diabetes models

• In vitro insulin resistance models: Hyperinsulinemic-hyperglycemic treatment conditions are used to create insulin-resistant cell models to study SMTNL1's insulin-sensitizing properties .

These models have revealed that SMTNL1 influences various pathways including progesterone signaling, myosin phosphatase regulation, and insulin receptor substrate phosphorylation.

What are the typical applications for SMTNL1 antibodies in research?

SMTNL1 antibodies serve as essential tools in multiple research applications:

• Western blotting: Used for semi-quantitative analysis of SMTNL1 protein expression and phosphorylation state (particularly at S301) in tissue and cell lysates .

• Immunofluorescence microscopy: Employed to visualize SMTNL1 localization and expression levels in cells and tissues, often combined with other markers to study protein interactions and cellular processes .

• High-content screening (HCS): Advanced microscopy technique used to quantitatively analyze SMTNL1's effects on cellular morphology, differentiation markers, and protein interactions in large sample sets .

• Protein-protein interaction studies: Used to investigate SMTNL1's interactions with binding partners such as MYPT1 (myosin phosphatase target subunit 1) and steroid hormone receptors including progesterone receptor (PR) .

• ELISA: Applied for quantitative detection of SMTNL1 in biological samples .

These applications have contributed to our understanding of SMTNL1's role in pregnancy-related adaptations and metabolic regulation.

How does SMTNL1 regulate insulin sensitivity at the molecular level?

SMTNL1 functions as an insulin-sensitizing agent through multiple molecular mechanisms:

• Regulation of IRS-1 phosphorylation: SMTNL1 reduces IRS-1 serine phosphorylation (particularly at Ser612) by downregulating ERK1/2 MAPK activity. This prevention of inhibitory phosphorylation maintains proper insulin signaling through the IRS-1-PI3K-Akt-mTOR pathway .

• Modulation of protein phosphatases: SMTNL1 increases the expression of specific protein phosphatases including PP2A and DUPS9, which contribute to decreased ERK1/2 activity and subsequently reduced IRS-1 inhibitory phosphorylation .

• Regulation of novel PKC isoforms: SMTNL1 downregulates the expression of novel type PKCε (nPKCε), which normally contributes to insulin resistance by promoting inhibitory phosphorylation of insulin signaling components .

• GLUT4 regulation: Evidence indicates that SMTNL1 can induce GLUT4-mediated glucose uptake in muscle cells, enhancing cellular glucose utilization .

These molecular mechanisms position SMTNL1 as a potential therapeutic target for insulin resistance and related metabolic disorders, particularly in the context of pregnancy and gestational diabetes.

What is the relationship between SMTNL1 and the myosin phosphatase (MP) complex in cellular function?

SMTNL1 has significant regulatory effects on the myosin phosphatase (MP) complex:

• Expression regulation: SMTNL1 reduces the expression of MYPT1, the regulatory subunit of MP, particularly under progesterone stimulation. This reduction was observed in both P4 (progesterone) and GDB (gestational diabetes model) conditions, with decreases of 39.49% and 50.91%, respectively, compared to control groups .

• Phosphorylation modulation: SMTNL1 overexpression significantly increases inhibitory phosphorylation of MYPT1 at Thr696, particularly in hyperglycemic/hyperinsulinemic conditions. This phosphorylation inhibits MP activity .

• Downstream effects on MLC20: While SMTNL1 does not affect MLC20 (20 kDa myosin light chain) expression levels, it influences MLC20 phosphorylation through MP regulation. This phosphorylation is critical for controlling cell contractility and migration .

• Functional consequences: By inhibiting MP activity, SMTNL1 promotes increased phosphorylation of contractile proteins, which contributes to reduced cell migration capacity, particularly in gestational diabetes models. This suggests a mechanism by which SMTNL1 may help maintain tissue integrity during metabolic stress .

The SMTNL1-MP interaction represents a key regulatory axis connecting hormonal signaling (particularly progesterone) with cytoskeletal dynamics and cell motility.

How does SMTNL1 function differ between pregnant and non-pregnant states?

SMTNL1 exhibits distinctive functions between pregnant and non-pregnant states:

In pregnancy, SMTNL1 works with progesterone receptor to promote a switch from oxidative to glycolytic metabolism in skeletal muscle, likely as an adaptation to meet the increased energy demands of pregnancy . Additionally, SMTNL1 helps regulate the physiological insulin resistance that naturally develops during pregnancy, balancing glucose availability for the developing fetus while maintaining maternal metabolic health .

Interestingly, smtnl1 −/− mice display impaired glucose tolerance, which is particularly pronounced during pregnancy, suggesting that SMTNL1 plays a crucial role in pregnancy-specific metabolic adaptations .

What are the critical factors to consider when selecting and validating SMTNL1 antibodies?

When selecting and validating SMTNL1 antibodies for research applications, consider these critical factors:

• Specificity: Ensure the antibody specifically recognizes SMTNL1 without cross-reactivity to related proteins. Validation should include positive controls (SMTNL1-overexpressing cells) and negative controls (smtnl1 −/− tissues) .

• Species reactivity: Confirm the antibody's reactivity with your species of interest. Available antibodies show reactivity with human, mouse, and rat SMTNL1 , but validation is necessary for each experimental system.

• Phospho-specificity: For studies of SMTNL1 phosphorylation (particularly at S301), use phospho-specific antibodies like anti-SMTNL1 S301A. Phosphorylation status can significantly impact SMTNL1 function .

• Application compatibility: Validate the antibody for your specific application (Western blot, immunofluorescence, etc.). The SMTNL1 Polyclonal Antibody (PACO13621) has been validated for Western blot and ELISA applications .

• Batch consistency: When conducting longitudinal studies, maintain consistency by using the same antibody lot when possible, as lot-to-lot variations can affect experimental outcomes.

• Sample preparation: Optimize sample preparation protocols (extraction buffers, fixation methods) to preserve SMTNL1 epitopes and ensure reliable detection.

Proper antibody validation using multiple techniques will enhance experimental reliability and facilitate accurate interpretation of results in SMTNL1 research.

What are the recommended protocols for detecting phosphorylated SMTNL1?

Detection of phosphorylated SMTNL1 requires specific methodological considerations:

• Sample preparation:

  • Add phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, and phosphatase inhibitor cocktails) to all lysis buffers

  • Maintain samples at 4°C during processing to minimize dephosphorylation

  • Use SDS-PAGE sample buffer with reducing agents to fully denature proteins

• Western blotting protocol:

  • Use phospho-specific antibodies like anti-SMTNL1 S301A for detecting specific phosphorylation sites

  • Include both phosphorylated and total SMTNL1 antibodies to calculate the phosphorylation ratio

  • Use Phos-tag™ acrylamide gels for enhanced separation of phosphorylated proteins

  • Include positive controls (e.g., SMTNL1 from progesterone-treated cells) and negative controls

• Immunofluorescence considerations:

  • Fix cells rapidly to preserve phosphorylation state (4% paraformaldehyde for 10-15 minutes)

  • Include phosphatase inhibitors in all buffers

  • Use high-affinity phospho-specific primary antibodies and sensitive detection systems

• Validation approaches:

  • Confirm phospho-antibody specificity using lambda phosphatase-treated samples as negative controls

  • Use site-directed mutants (e.g., S301A) to validate phospho-site specificity

  • Compare results across multiple detection methods (Western blot, immunofluorescence)

These protocols will facilitate accurate detection and quantification of SMTNL1 phosphorylation, which is critical for understanding its functional regulation in different physiological contexts.

How can I troubleshoot weak or non-specific signals in SMTNL1 Western blots?

When encountering weak or non-specific signals in SMTNL1 Western blots, consider these troubleshooting strategies:

• For weak signals:

  • Increase protein loading (30-50 μg total protein)

  • Optimize primary antibody concentration and incubation conditions (try overnight at 4°C)

  • Enhance detection sensitivity using high-sensitivity chemiluminescent substrates

  • Consider protein enrichment techniques (immunoprecipitation) for low-abundance samples

  • Use fresh samples and avoid repeated freeze-thaw cycles

• For non-specific bands:

  • Increase blocking stringency (5% BSA or milk, longer blocking time)

  • Optimize antibody dilution (typically 1:500-1:2000 range for SMTNL1 antibodies)

  • Include additional washing steps with higher detergent concentration

  • Validate specificity using smtnl1 −/− tissues as negative controls

  • Pre-absorb antibody with recombinant SMTNL1 protein to confirm specificity

• Technical optimization:

  • Adjust transfer conditions for efficient protein transfer (particularly important for higher MW proteins)

  • Use freshly prepared buffers and reagents

  • Consider gradient gels for better resolution of SMTNL1 and potential post-translationally modified forms

  • For phospho-specific detection, ensure phosphatase inhibitors are present throughout sample preparation

• Antibody selection considerations:

  • Try alternative SMTNL1 antibody clones if available

  • Verify the antibody recognizes the appropriate species and isoform

  • For human samples, confirm the antibody recognizes the specific SMTNL1 variants present in your tissue/cell type

These troubleshooting approaches should help resolve common issues with SMTNL1 Western blotting and improve experimental reproducibility.

What are common pitfalls when studying SMTNL1 in pregnancy and metabolic disease models?

Researchers studying SMTNL1 in pregnancy and metabolic disease models should be aware of these common pitfalls:

• Timing considerations:

  • Pregnancy stage significantly affects SMTNL1 expression and function; precise documentation of gestational day is critical (days 14-17 are commonly used in mouse studies)

  • Hormonal fluctuations during estrous cycle in non-pregnant females can cause data variability; cycle stage should be determined and controlled

• Model-specific challenges:

  • In knockout models, compensatory mechanisms may mask SMTNL1-dependent phenotypes

  • Cell culture models may not fully recapitulate the complex hormonal environment of pregnancy

  • Gestational diabetes models require careful standardization of glucose and insulin levels to ensure reproducibility

• Interpretation pitfalls:

  • SMTNL1 effects differ between tissues (skeletal muscle vs. endometrium); tissue-specific analyses are necessary

  • Correlation between mRNA and protein levels may be poor; both should be measured

  • Phosphorylation state critically affects SMTNL1 function and should be assessed alongside total protein levels

• Technical challenges:

  • Low endogenous SMTNL1 expression in some tissues may require sensitive detection methods

  • SMTNL1 interacts with multiple signaling pathways (progesterone receptor, myosin phosphatase); pathway-specific controls are needed to interpret results accurately

  • Metabolic phenotypes are influenced by multiple factors (diet, age, genetic background); these variables must be carefully controlled

• Translational considerations:

  • Mouse models may not fully reflect human physiology; validation in human samples or cell lines is recommended

  • The 4% prevalence of gestational diabetes mentioned in literature highlights the clinical relevance but also the heterogeneity of this condition

Awareness of these pitfalls will improve experimental design and interpretation when studying SMTNL1 in pregnancy and metabolic disease contexts.

What are promising therapeutic applications for targeting SMTNL1 in metabolic disorders?

Based on current understanding of SMTNL1 biology, several promising therapeutic applications emerge:

• Gestational diabetes management:

  • SMTNL1 activation or mimetics could potentially attenuate insulin resistance during pregnancy

  • Targeted approaches to enhance endometrial SMTNL1 activity may reduce complications of gestational diabetes

  • Diagnostic applications measuring SMTNL1 levels or activation state could identify at-risk pregnancies

• Insulin sensitization strategies:

  • SMTNL1's ability to enhance insulin sensitivity through reduced IRS-1 inhibitory phosphorylation suggests therapeutic potential beyond pregnancy

  • Small molecules enhancing SMTNL1-progesterone receptor interactions could provide tissue-specific insulin sensitization

  • SMTNL1-derived peptides targeting specific protein-protein interactions could modulate metabolic pathways

• Muscle metabolism modulation:

  • SMTNL1's role in muscle fiber-type switching suggests applications in conditions requiring metabolic adaptation

  • Therapeutic approaches activating SMTNL1 pathways could potentially enhance glycolytic metabolism in specific contexts

  • Exercise-mimetic effects might be achievable through targeted SMTNL1 pathway modulation

• Reproductive health applications:

  • SMTNL1's effects on endometrial epithelial cell differentiation suggest potential applications in reproductive medicine

  • Modulation of SMTNL1 activity could potentially address endometrial dysfunction associated with metabolic disorders

  • Fertility applications given the reproductive phenotypes observed in smtnl1 −/− mice

Research challenges include developing specific modulators of SMTNL1 function, understanding tissue-specific effects, and translating findings from animal models to human applications. Continued investigation of SMTNL1's regulation and downstream effects will be crucial for realizing these therapeutic possibilities.

How can advanced imaging techniques enhance our understanding of SMTNL1 function?

Advanced imaging approaches offer powerful opportunities to expand our understanding of SMTNL1 biology:

• High-content screening microscopy:

  • The research already demonstrates successful application of HCS to study SMTNL1's effects on endometrial cell differentiation and migration

  • Multiplexed imaging with automated analysis can reveal subtle phenotypes across large sample sets

  • Quantitative assessment of multiple parameters simultaneously (protein expression, phosphorylation, localization, morphology) provides comprehensive data

• Live-cell imaging applications:

  • FRET-based biosensors could monitor SMTNL1 interactions with binding partners (PR, MYPT1) in real-time

  • Fluorescently-tagged SMTNL1 would enable tracking of subcellular localization dynamics in response to hormones or metabolic changes

  • Optogenetic approaches could enable precise temporal control of SMTNL1 function to dissect signaling kinetics

• Super-resolution microscopy:

  • Techniques such as STORM or PALM could reveal nanoscale organization of SMTNL1 with interacting proteins

  • Two-color super-resolution imaging could map precise spatial relationships between SMTNL1 and components of insulin signaling or myosin phosphatase complexes

  • Quantitative single-molecule localization could determine stoichiometry of SMTNL1 complexes

• In vivo imaging possibilities:

  • Development of SMTNL1 reporter mice would enable longitudinal studies of expression during pregnancy

  • Tissue-clearing techniques combined with light-sheet microscopy could map SMTNL1 expression across intact organs

  • Intravital microscopy could potentially monitor SMTNL1-dependent processes in real-time in living animals

These advanced imaging approaches would complement biochemical and molecular studies to provide spatiotemporal information about SMTNL1 function, particularly in dynamic processes such as pregnancy-induced adaptations and cellular responses to metabolic stress.