CRTC1 Monoclonal Antibody

What is CRTC1 and why is it significant in research?

CRTC1 is a cytoplasmic coactivator that translocates to the nucleus in response to cAMP signaling, where it augments CREB (cAMP-response element-binding protein) activity to promote transcription of target genes. CRTC1 is the most abundant CRTC isoform in neuronal cells and plays crucial roles in metabolism, energy expenditure, and cellular proliferation. Research significance stems from CRTC1's involvement in obesity, cancer development, and aging-related metabolic changes . Unlike other transcriptional regulators, CRTC1 has a distinctive mechanism where it binds directly to the bZIP domain of transcription factors like CREB, c-Jun, and c-Fos, making it an interesting target for studying transcriptional regulation mechanisms.

What cell types and tissues express CRTC1 at detectable levels?

CRTC1 expression has been validated in multiple cell types and tissues. Based on antibody testing data, CRTC1 protein is detectable in mouse kidney tissue, brain tissue, and multiple cell lines including A431, Jurkat, and L02 cells . In the brain, CRTC1 is particularly abundant in neuronal cells, with high expression in melanocortin-4 receptor (MC4R)-expressing neurons that regulate appetite and energy metabolism . When designing experiments, researchers should consider these expression patterns to select appropriate positive controls.

What are the recommended positive controls for CRTC1 antibody validation?

For CRTC1 antibody validation, several reliable positive controls have been documented:

• Mouse brain tissue (particularly for neuronal CRTC1 studies)

• Mouse kidney tissue

• Human cell lines: A431, Jurkat, and L02 cells

These samples consistently show CRTC1 expression and can be used to validate antibody specificity and sensitivity. When establishing a new experimental system, researchers should first confirm CRTC1 detection in these known positive samples before proceeding to experimental samples.

What are the validated applications for CRTC1 monoclonal antibodies?

Based on reported testing data, CRTC1 monoclonal antibodies have been successfully used in the following applications:

When using CRTC1 antibodies for ChIP applications, researchers have successfully detected CRTC1 binding to promoters of genes like MMP1 following stimulation with agonists such as TPA .

How should samples be prepared for optimal CRTC1 detection by Western blot?

For optimal detection of CRTC1 in Western blot applications:

• Complete lysis buffer should contain phosphatase inhibitors, as CRTC1 undergoes significant post-translational modifications that affect its mobility on gels

• CRTC1 typically appears as a diffuse band in total cell lysates, but a discrete band with slower mobility is observed when associated with AP-1 transcription factors

• When analyzing nuclear translocation, separate nuclear and cytoplasmic fractions should be prepared using nuclear/cytoplasmic extraction protocols

• Samples should be freshly prepared when possible, as CRTC1 may be subject to degradation during prolonged storage

Researchers should note that CRTC1's apparent molecular weight may vary depending on its phosphorylation state, which changes in response to cellular stimuli like cAMP activation.

What are the common troubleshooting issues with CRTC1 antibodies and how can they be addressed?

When working with CRTC1 antibodies, researchers may encounter several technical challenges:

• Diffuse banding patterns: This is characteristic of CRTC1 due to post-translational modifications. Consider using phosphatase treatments to resolve this issue when studying non-phosphorylated forms.

• Cross-reactivity with CRTC2/CRTC3: Due to sequence homology between CRTC isoforms, verify antibody specificity using CRTC1-knockout samples or siRNA-treated cells.

• Weak nuclear signals: When studying nuclear translocation, pretreat cells with stimuli known to induce translocation (cAMP activators like forskolin) as positive controls to ensure the detection system works.

• Inconsistent co-immunoprecipitation results: Add protease and phosphatase inhibitors to preserve protein-protein interactions, and consider crosslinking approaches for transient interactions.

How can CRTC1 antibodies be used to study its interaction with different transcription factors?

CRTC1 interacts with multiple transcription factors beyond CREB, including c-Jun and c-Fos of the AP-1 complex. To study these distinct interactions:

• Co-immunoprecipitation experiments: CRTC1 antibodies can be used to pull down protein complexes, followed by Western blotting for different transcription factor partners. Research has shown that CRTC1 associates with both c-Jun and c-Fos through direct binding of its N-terminal region (amino acids 1-142) to their bZIP domains .

• Sequential ChIP (Re-ChIP): For studying promoter-specific complexes, perform ChIP with CRTC1 antibodies followed by a second round of immunoprecipitation with antibodies against specific transcription factors to identify co-occupancy at particular genomic loci.

• Proximity ligation assays: To visualize interactions in intact cells, combine CRTC1 antibodies with antibodies against potential binding partners in this technique that produces fluorescent signals only when proteins are in close proximity.

These approaches have revealed that CRTC1 forms different complexes that regulate distinct gene sets - CREB-dependent genes like EVX1 versus AP-1-dependent genes like MMP1, MTIIA, and TIMP1 .

What experimental designs are recommended for studying CRTC1's role in metabolic disorders?

When investigating CRTC1's function in metabolic regulation:

• Age-dependent studies: Research has shown that CRTC1's effects on dietary fat intake become more pronounced with age. Design longitudinal studies that track metabolic parameters from youth to advanced age in models with CRTC1 manipulation .

• Tissue-specific knockouts: Since whole-body CRTC1 knockout produces complex phenotypes, use conditional knockouts (such as MC4R neuron-specific CRTC1 deletion) to dissect tissue-specific functions .

• Diet challenges: Compare normal chow versus high-fat diets, as CRTC1-deficient models may only develop obesity under high-fat dietary conditions. Studies have shown that MC4R neuron-specific CRTC1-knockout mice become obese on high-fat diet but not on normal chow .

• Leptin sensitivity testing: Assess leptin signaling, as CRTC1 deficiency has been linked to leptin resistance. Measure phosphorylated STAT3 levels following leptin administration to determine signaling efficiency .

• Comparison of different dietary fats: Research indicates differential effects of distinct fat sources (soybean oil versus lard) on CRTC1-deficient phenotypes, suggesting experimental designs should test multiple fat sources .

How can CRTC1 antibodies be utilized to investigate its role in cell proliferation and cancer?

CRTC1 has significant roles in cell proliferation and oncogenesis, particularly through its interaction with AP-1 transcription factors and as part of the CRTC1-Maml2 fusion oncoprotein. Research approaches include:

• Colony formation assays: Measure the effect of CRTC1 manipulation (overexpression, knockdown) on c-Jun-dependent cellular proliferation. Studies have demonstrated that CRTC1 synergizes with c-Jun to promote cellular growth, while AP-1-dependent proliferation is abrogated in CRTC1-deficient cells .

• BrdU incorporation assays: Quantify active cell cycling in the presence or absence of CRTC1. Research has shown that c-Jun-induced cell proliferation is completely abolished in CRTC1-deficient cells .

• Chromatin occupancy studies: Use ChIP with CRTC1 antibodies to identify genomic targets following mitogenic stimulation (e.g., TPA treatment). CRTC1 and c-Jun show coincident occupancy at promoters like MMP1 in response to TPA .

• Fusion protein studies: For mucoepidermoid carcinomas, investigate the CRTC1-Maml2 oncoprotein using antibodies that recognize the CRTC1 portion to understand how this fusion activates both c-Jun and c-Fos to drive cellular transformation .

What considerations are important when designing experiments to study CRTC1 activation?

CRTC1 activation involves translocation from the cytoplasm to the nucleus following dephosphorylation. Key experimental design considerations include:

• Stimulation conditions: Use appropriate stimuli known to activate CRTC1:

  • cAMP-elevating agents (forskolin/IBMX) for CREB-dependent pathways

  • TPA (phorbol ester) for AP-1-dependent pathways

• Time course experiments: CRTC1 translocation occurs rapidly following stimulation, typically within 15-30 minutes. Design time course experiments to capture both immediate and sustained responses.

• Subcellular fractionation: Properly separate nuclear and cytoplasmic fractions to quantify translocation efficiency.

• Phosphorylation state analysis: Use phospho-specific antibodies or phosphatase treatments to correlate dephosphorylation with nuclear translocation and activity.

• Downstream target measurement: Assess appropriate target genes based on the pathway being studied:

  • CREB pathway: EVX1

  • AP-1 pathway: MMP1, MTIIA, TIMP1

How should researchers interpret conflicting data regarding CRTC1 function in different experimental models?

When facing contradictory results across different CRTC1 studies:

• Consider the specific CRTC1 functional domain being studied. The N-terminal region (amino acids 1-142) binds transcription factors, while other regions have different functions .

• Evaluate the experimental context, as CRTC1 functions differently across tissues. For example, CRTC1 deficiency in MC4R neurons shows age-dependent phenotypes that may not be apparent in younger animals .

• Compare diet conditions across studies. Some CRTC1 phenotypes only manifest under specific dietary challenges, such as high-fat diets versus normal chow .

• Assess the method of dietary fat administration, as spontaneous fat ingestion may yield different results compared to forced high-fat diet consumption .

• Consider compensatory mechanisms from other CRTC family members (CRTC2, CRTC3) that may mask phenotypes in certain models.

What are the key methodological considerations when studying age-dependent functions of CRTC1?

Research has revealed that CRTC1's regulatory effects on dietary fat intake increase with age . When designing age-related CRTC1 studies:

• Include multiple age groups within the same experiment (e.g., young adults at 6-10 weeks, middle-aged at 20-30 weeks, and older animals at 40+ weeks).

• Measure parameters consistently across all age groups, including:

  • Body weight and composition

  • Food intake (separated by macronutrient when possible)

  • Energy expenditure

  • Glucose metabolism (fasting glucose, glucose tolerance tests)

  • Gene expression profiles in relevant tissues

• Consider tissue collection at multiple time points to track molecular changes preceding phenotypic manifestations.

• Use age-matched controls for all experimental groups, as baseline metabolism changes with age independently of CRTC1 manipulation.

• Account for hormonal changes that occur with aging, as these may interact with CRTC1 signaling pathways.

What are promising research areas for CRTC1 antibody applications in neuroscience?

Given CRTC1's significant expression in neuronal tissues and its involvement in metabolic regulation, several promising research directions emerge:

• Circuit-specific CRTC1 function: Use CRTC1 antibodies in combination with neural circuit tracing to identify specific neuronal populations where CRTC1 regulates feeding behavior and energy expenditure beyond the established MC4R-expressing neurons .

• Synaptic plasticity: Investigate CRTC1's role in learning and memory through its regulation of CREB-dependent gene expression in response to neuronal activity.

• Neurodegenerative diseases: Explore CRTC1 dysfunction in conditions like Alzheimer's disease, where metabolic dysregulation contributes to pathology.

• Aging brain: Study how age-dependent changes in CRTC1 function affect neuronal health and cognitive function, given the established link between CRTC1 function and aging .

• Stress responses: Investigate how stress signals modulate CRTC1 activity in key brain regions related to both metabolic and behavioral adaptation.

How might CRTC1 antibodies contribute to therapeutic target validation in metabolic disorders?

CRTC1 antibodies can facilitate target validation for metabolic disorders through:

• Biomarker development: Establish whether CRTC1 phosphorylation state or localization correlates with disease progression in obesity models.

• Target engagement studies: For compounds designed to modulate CRTC1 activity, use antibodies to verify whether the compound affects CRTC1 phosphorylation, localization, or complex formation.

• Pathway analysis: Identify downstream effectors and feedback mechanisms in the CRTC1 signaling network that might serve as alternative intervention points.

• Age-appropriate interventions: Given that CRTC1's metabolic effects intensify with age, use antibodies to track age-dependent changes that might inform timing of therapeutic interventions .

• Patient stratification: Develop immunoassays to identify patients with altered CRTC1 activity who might benefit from targeted therapies.