CRTC3 Antibody

What is CRTC3 and why is it significant in cellular signaling research?

CRTC3 functions as a co-activator for CREB (cAMP response element-binding protein), playing essential roles in various signaling pathways. It serves as a key sensor and regulator for melanogenesis, orchestrating this process by directly targeting microphthalmia-associated transcription factor (MITF) and regulating the expression of melanogenesis-related genes . Additionally, CRTC3 is activated in response to catecholamine signals and contributes to metabolic regulation by modulating adenyl cyclase activity through upregulation of RGS2 . In brown adipose tissue (BAT), CRTC3 reduces function by down-regulating sympathetic nerve activity and vascularization, influencing thermogenesis and energy homeostasis .

In which tissues should researchers expect significant CRTC3 expression?

Unlike CRTC1, which is expressed primarily in brain tissue, CRTC3 protein and mRNA amounts are particularly abundant in white adipose tissue and, to a lesser extent, in brown adipose tissue . This tissue-specific expression pattern is important for researchers to consider when designing experiments involving CRTC3 antibodies. Studies have also demonstrated CRTC3 expression in melanocytes, where it plays a crucial role in pigmentation pathways . When planning immunohistochemistry or tissue analysis experiments, researchers should consider these expression patterns for proper experimental controls.

What are the recommended antibody validation approaches for CRTC3 detection?

For rigorous CRTC3 antibody validation, researchers should:

• Perform Western blotting with positive controls (tissues known to express CRTC3, such as white adipose tissue) and negative controls (CRTC3 knockout tissues where available)

• Include peptide competition assays to confirm specificity

• Validate using multiple antibodies targeting different epitopes of CRTC3

• Test specificity against other CRTC family members (CRTC1, CRTC2) to confirm lack of cross-reactivity

• Consider siRNA or CRISPR-mediated knockdown of CRTC3 to confirm antibody specificity

Knockout validation is particularly valuable, as demonstrated in studies using CRTC3-null mice with deletion of exon 1 (encoding the CREB Binding Domain) .

What are the optimal protocols for detecting CRTC3 via immunofluorescence in tissue sections?

For effective immunofluorescence detection of CRTC3 in tissue sections, the following protocol has been validated in research settings:

• Perform antigen retrieval using pressure cooking (120.5°C for 30 seconds followed by 90°C for 10 seconds) in antigen unmasking solution

• Block sections appropriately to reduce background

• Incubate with primary CRTC3 antibody at 1:100 dilution at 4°C overnight

• Detect using FITC-conjugated anti-mouse secondary antibody (1:500) at 4°C for 30 minutes

• For co-localization studies with melanocyte markers, anti-rabbit Alexa Fluor 546 (1:500) can be used to detect Pmel and MLANA

• Acquire images using confocal microscopy for optimal resolution

This protocol has been successfully employed to visualize CRTC3 localization in relation to melanocyte-specific markers in skin tissue sections.

How should subcellular localization of CRTC3 be monitored in response to stimuli?

CRTC3 translocation between cytoplasm and nucleus is a key regulatory mechanism affected by cAMP signaling. To monitor this process:

• Generate CRTC3-EGFP fusion plasmids for live-cell imaging

• Transfect target cells (e.g., B16F10 melanoma cells) using appropriate transfection reagents like PEI

• After 24 hours of transfection, treat cells with activators of interest (e.g., forskolin [FSK] or TPA)

• Monitor subcellular localization using fluorescence microscopy

• For fixed-cell analysis, use immunofluorescence with CRTC3 antibodies and nuclear counterstains

Research has shown that short-term (0.5-1 hour) exposure to cAMP agonists promotes dephosphorylation and nuclear entry of CRTC3, while prolonged cAMP stimulation triggers CRTC3 degradation .

What controls are essential when using CRTC3 antibodies in Western blotting applications?

When performing Western blotting with CRTC3 antibodies, include:

• Positive tissue controls: white adipose tissue samples (high expression)

• Negative controls: CRTC3 knockout samples where available

• Loading controls: α-tubulin (1:5,000) or HSP90 have been validated

• Phosphorylation-state controls: when studying CRTC3 activation, include total CREB and phospho-CREB antibodies for pathway verification

• Secondary antibody controls: goat anti-rabbit IgG-HRP (1:5,000) or goat anti-mouse IgG-HRP (1:5,000) depending on the primary antibody host species

For enhanced specificity confirmation, use two different CRTC3 antibodies targeting distinct epitopes.

How can CRTC3 antibodies be employed to study melanogenesis regulatory mechanisms?

For investigating CRTC3's role in melanogenesis:

• Use CRTC3 antibodies in combination with melanocyte markers (MITF, tyrosinase, Tyrp1, DCT) in co-immunoprecipitation studies to examine protein-protein interactions

• Employ chromatin immunoprecipitation (ChIP) with CRTC3 antibodies to identify direct genomic targets in melanocytes

• Combine with MITF antibodies to study coordinated regulation of pigmentation genes

• Integrate with phospho-specific antibodies to monitor CRTC3 activation state in response to UVB or cAMP stimulation

• Study CRTC3 localization in relation to melanin production using melanin assays in parallel

Research has demonstrated that CRTC3 directly targets MITF and regulates most key melanogenesis-related genes, making it an important focus for pigmentation studies .

What methodological approaches are recommended for studying CRTC3's role in metabolic regulation?

To investigate CRTC3 in metabolic contexts:

• Combine CRTC3 immunodetection with metabolic pathway analysis in adipose tissues

• Monitor CRTC3 phosphorylation state in response to catecholamines using phospho-specific antibodies

• Assess CRTC3-dependent gene expression using antibodies for downstream targets like RGS2

• Compare CRTC3 activity between different fat depots (white vs. brown adipose tissue)

• In studies of insulin sensitivity, correlate CRTC3 activation with insulin signaling pathway components

Studies have shown that CRTC3-null mice have 50% lower adipose tissue mass despite comparable food intake and physical activity to control mice, indicating CRTC3's significant metabolic functions .

How can CRTC3 antibodies be integrated into cancer research protocols?

For melanoma and cancer research applications:

• Use CRTC3 antibodies to assess expression levels in patient-derived samples via tissue microarrays

• Compare CRTC3 subcellular localization between normal melanocytes and melanoma cells

• Investigate correlation between CRTC3 expression/activity and patient outcomes

• Study CRTC3 in relation to BRAF inhibitor resistance mechanisms

• Monitor interaction between CRTC3 and ERK1/2 signaling pathways using co-immunoprecipitation

Research has shown that alterations of CRTC3 were observed in 10% of melanoma samples in the TCGA database, with most alterations increasing CRTC3 expression and being associated with reduced patient survival .

How should researchers interpret contradictory results between CRTC3 protein levels and activity?

CRTC3 activity is primarily regulated by post-translational modifications rather than expression levels alone. When facing contradictory results:

• Assess phosphorylation status of CRTC3, as this determines its activity and localization

• Examine subcellular localization (nuclear vs. cytoplasmic) as a measure of activity

• Evaluate downstream target gene expression (e.g., OCA2, PDE4B, PDE4D) as functional readouts

• Consider interactions with CREB and other transcriptional machinery components

• Account for potential compensatory mechanisms from other CRTC family members

For example, studies have shown that low forskolin concentrations (800nM) increased nuclear amounts of CRTC3 to a greater extent than CRTC1 or CRTC2, indicating differential sensitivity to cAMP levels .

What are the common technical challenges with CRTC3 antibodies and how can they be addressed?

Common challenges include:

• Cross-reactivity with other CRTC family members: Validate using CRTC3 knockout samples and peptide competition assays

• Variable detection of phosphorylated forms: Use phospho-specific antibodies when available; alternatively, use Phos-tag gels to separate phosphorylated forms

• Inconsistent immunoprecipitation efficiency: Optimize buffer conditions and antibody concentrations; test multiple antibodies targeting different epitopes

• Background in tissue sections: Implement stringent blocking procedures and include appropriate negative controls

• Degradation during sample preparation: Include protease and phosphatase inhibitors in all buffers to preserve CRTC3 integrity

When troubleshooting, remember that CRTC3 undergoes degradation after prolonged cAMP stimulation, which can affect detection in stimulated samples .

What experimental design considerations are important when studying CRTC3's role in pigmentation disorders?

When investigating pigmentation disorders:

• Compare CRTC3 expression, phosphorylation, and localization between normal and affected tissue samples

• Develop co-culture systems between melanocytes and keratinocytes to study CRTC3-dependent paracrine regulation

• Use CRTC3 antibodies in combination with melanocyte markers to assess cell-specific alterations

• Implement ex vivo human skin models for translational relevance

• Consider genetic background effects when using animal models, as pigmentation pathways vary across species

Research has shown that CRTC3 can serve as a screening tool for discovering melanogenesis-modulating small molecules, making it valuable for pigmentation disorder research .

How can CRTC3 antibodies be used to investigate thermogenesis in brown adipose tissue?

For BAT thermogenesis studies:

• Monitor CRTC3 expression and activation state in response to cold exposure or β-adrenergic stimulation

• Compare CRTC3 localization patterns between BAT of cold-acclimatized and control animals

• Investigate correlation between CRTC3 activity and expression of thermogenic genes (UCP1, PGC1α)

• Use CRTC3 antibodies to study tissue-specific knockout models (e.g., UCP1-Cre;CRTC3-flox)

• Assess CRTC3's relationship with sympathetic innervation and vascularization markers in BAT

Studies have demonstrated that CRTC3 inhibits BAT activity by disrupting the expression of neurotrophins and proangiogenic factors that otherwise promote sympathetic innervation and vascularization of BAT, highlighting its importance in energy homeostasis .