CREBL2 Antibody

What is CREBL2 and why is it important in research?

CREBL2 (cAMP Responsive Element Binding Protein Like 2) is a protein coding gene that functions as a probable regulator of CREB1 transcriptional activity involved in adipose cell differentiation and metabolic regulation. It shares 41% identity with CRE-binding protein (CREB) over a 48-base region encoding the bZip domain, suggesting DNA binding capabilities. CREBL2 is particularly significant in research because it's located in a chromosomal region (12p13) frequently deleted in hematopoietic malignancies, breast, non-small-cell lung, and ovarian cancers, suggesting it may function as a tumor suppressor gene . Additionally, recent studies have identified CREBL2 as a key regulator of cellular metabolism, linking nutrient sensing via mTORC1 to metabolic responses in cells .

How do I choose the appropriate CREBL2 antibody for my research application?

When selecting a CREBL2 antibody, consider:

• Experimental application requirements (Western blot, ELISA, immunofluorescence)

• Species reactivity needed (human, mouse, rat)

• Antibody type (polyclonal vs. monoclonal)

For most metabolic studies examining CREBL2 function, researchers should select antibodies validated for Western blot applications with demonstrated specificity. Commercial polyclonal antibodies with reactivity against human, mouse, and rat CREBL2 are available with validation for ELISA and Western blot . When studying protein-protein interactions (such as CREBL2-Crebrf binding), ensure the epitope recognized doesn't interfere with binding domains. For quantitative applications, consider antibodies with demonstrated linear response curves in the concentration range of interest.

What are the typical technical specifications of CREBL2 antibodies?

Most commercially available CREBL2 antibodies have the following specifications:

The antibody is typically supplied in PBS buffer (pH 7.3) with 0.02% sodium azide and 50% glycerol .

How should I design knockdown experiments to study CREBL2 function?

When designing knockdown experiments to study CREBL2 function, implement the following methodology:

• Select appropriate cell lines relevant to your metabolic pathway of interest (e.g., C2C12 myoblasts for muscle metabolism or Hepa1-6 for hepatic function)

• Design at least two independent siRNAs targeting different regions of CREBL2 mRNA to control for off-target effects

• Include appropriate controls (non-targeting siRNA)

• Validate knockdown efficiency using both RT-qPCR and Western blot

• Plan comprehensive metabolic phenotyping including:

  • Triglyceride content analysis

  • Glucose uptake measurements

  • Glycolysis assessment (e.g., lactate secretion)

  • Transcriptional profiling (RNA-Seq)

Research has shown that Crebl2 knockdown leads to significant increases in triglyceride content in both C2C12 myoblasts and Hepa1-6 cells, demonstrating its role in lipid metabolism regulation .

What methods are most effective for studying CREBL2 transcriptional targets?

To effectively study CREBL2 transcriptional targets:

• Perform RNA-Seq after CREBL2 knockdown in relevant cell types. Previous studies in C2C12 cells identified 408 genes downregulated and 164 genes upregulated upon Crebl2 knockdown .

• Validate key targets using RT-qPCR on independent biological replicates.

• Conduct pathway analysis on differentially expressed genes - both up and downregulated genes were enriched in glycoproteins and proteins with signal peptides.

• For direct binding assessment, perform ChIP-seq experiments using validated CREBL2 antibodies.

• Functional validation of key targets through individual knockdown experiments. For example, knockdown of 7 out of 9 genes downregulated by Crebl2 knockdown showed a tendency to increase cellular TAG levels .

This multi-layered approach allows for comprehensive identification of both direct and indirect CREBL2 transcriptional targets.

How can I investigate CREBL2's role in the mTORC1 signaling pathway?

To investigate CREBL2's role in the mTORC1 signaling pathway:

• Treat cells with rapamycin (mTORC1 inhibitor) in the presence or absence of CREBL2 knockdown.

• Perform genome-wide expression analysis to identify genes induced by rapamycin treatment in control cells compared to CREBL2-knockdown cells.

• Focus on genes that show blunted induction upon CREBL2 knockdown, as these represent mTORC1-responsive genes that require CREBL2 for appropriate activation .

• Validate key targets using RT-qPCR, focusing on:

  • Genes not affected at baseline by CREBL2 knockdown but losing rapamycin induction (e.g., Bloc1s1, Fbxo36)

  • Genes with elevated baseline expression after CREBL2 knockdown (e.g., Cxcl12, Ing4)

• Analyze Crebrf expression, which is induced by rapamycin treatment in multiple cell types .

This approach has successfully identified CREBL2 as mediating part of the transcriptional response caused by mTORC1 inhibition.

How does CREBL2 interact with Crebrf to regulate cellular metabolism?

The CREBL2-Crebrf interaction represents a critical regulatory complex in cellular metabolism:

• Co-immunoprecipitation experiments demonstrate that myc-tagged CREBL2 can bind HA-tagged Crebrf, forming a transcriptional complex similar to their Drosophila orthologs REPTOR and REPTOR-BP .

• This interaction appears stable regardless of mTORC1 activity, as rapamycin treatment does not affect the amount of Crebrf co-immunoprecipitating with Crebl2 .

• The subcellular localization of this complex is variable - epitope-tagged Crebrf can be nuclear, cytoplasmic, or both in TSC2−/− MEFs, while in HEK293T cells, HA-Crebrf is mostly cytoplasmic .

• Functionally, this complex regulates metabolic gene expression downstream of mTORC1 signaling.

• Crebrf is induced by rapamycin treatment in multiple cell types (TSC2−/− MEFs, C2C12 myoblasts), suggesting a general role of the Crebrf/Crebl2 complex upon mTORC1 inhibition .

To study this interaction in your research, combine co-IP approaches with subcellular fractionation and transcriptional reporter assays to determine how environmental conditions alter complex formation and activity.

What are the implications of CREBL2's potential tumor suppressor role for cancer research?

CREBL2's potential tumor suppressor function has significant implications for cancer research:

• CREBL2 was identified in a commonly deleted region on chromosome 12p13 flanked by ETV6 and CDKN1B genes, associated with hematopoietic malignancies, breast, non-small-cell lung, and ovarian cancers .

• Its DNA-binding capabilities (via the bZip domain) suggest it may regulate genes involved in cell cycle control or metabolism.

• Research approaches should include:

  • Analysis of CREBL2 expression across cancer types using public databases (TCGA, CCLE)

  • Correlation of CREBL2 loss with clinical outcomes

  • Functional studies in cancer cell lines with CREBL2 reintroduction

  • Investigation of metabolic alterations in cancer cells with CREBL2 deletion (given its established role in regulating cellular metabolism)

  • Examination of potential synthetic lethal interactions in CREBL2-deficient cells

Given that CREBL2 knockdown increases triglyceride content in cells , researchers should explore whether this metabolic alteration contributes to the cancer phenotype of cells with CREBL2 deletion.

How can genome-wide approaches be used to understand CREBL2's regulatory network?

To comprehensively map CREBL2's regulatory network:

• Integrate multiple genome-wide datasets:

  • RNA-Seq following CREBL2 knockdown (identified 408 genes downregulated and 164 genes upregulated in C2C12 cells; 267 genes downregulated and 396 genes upregulated in Hepa1-6 cells)

  • ChIP-Seq to identify direct CREBL2 binding sites genome-wide

  • ATAC-Seq to assess chromatin accessibility changes following CREBL2 manipulation

  • Metabolomics to correlate transcriptional changes with metabolic alterations

• Apply computational approaches:

  • Motif analysis of CREBL2 binding sites to define consensus sequences

  • Pathway enrichment analysis (both upregulated and downregulated genes show enrichment for glycoproteins and proteins with signal peptides)

  • Integration with other transcription factor binding data to identify cooperative or antagonistic relationships

• Validate key nodes in the network through targeted approaches:

  • Single gene knockdowns to assess phenotypic impacts (previous studies showed that knocking down 7 out of 9 genes downregulated by Crebl2 knockdown tended to increase cellular TAG levels)

  • Reporter assays for direct transcriptional regulation

This multi-omics approach will provide a systems-level understanding of CREBL2's regulatory role in cellular metabolism.

Why might I observe inconsistent results when using CREBL2 antibodies for immunofluorescence?

Inconsistent immunofluorescence results with CREBL2 antibodies may occur for several reasons:

• Antibody specificity issues: Multiple commercial antibodies for CREBL2 have failed to give specific signals in MEFs, Hepa1-6 cells, or C2C12 cells . Even experienced researchers have reported difficulties generating specific antibodies against CREBL2.

• Variable subcellular localization: Research has shown that epitope-tagged CREBL2 can exhibit variable localization patterns. HA-tagged Crebrf (which interacts with CREBL2) was found to be nuclear, cytoplasmic, or both in TSC2−/− MEFs, varying from cell to cell .

• Technical solutions:

  • Use epitope-tagged CREBL2 constructs as positive controls

  • Include CREBL2 knockdown samples as negative controls

  • Optimize fixation protocols (test both paraformaldehyde and methanol fixation)

  • Validate antibody specificity by Western blot before immunofluorescence

  • Consider cell type-specific optimization, as localization may vary between cell types

If consistent results cannot be achieved with antibodies, consider using fluorescently tagged CREBL2 constructs for localization studies.

What controls should I include when using CREBL2 antibodies for Western blotting?

When using CREBL2 antibodies for Western blotting, include these essential controls:

• Positive control: Lysate from cells known to express CREBL2 (C2C12, Hepa1-6, or HEK293T cells transfected with CREBL2 expression construct)

• Negative control: Lysate from cells with CREBL2 knockdown (siRNA or shRNA)

• Loading control: Probe for housekeeping proteins (β-actin, GAPDH, or α-tubulin)

• Molecular weight marker: Ensure the detected band matches the expected size (17 kDa)

• Antibody validation controls:

  • Peptide competition assay (pre-incubating antibody with immunizing peptide should abolish specific signal)

  • Test multiple antibody dilutions (recommended range: 1/500 - 1/2000)

  • Include both reducing and non-reducing conditions if investigating complex formation

Additionally, optimize protein extraction protocols as transcription factors like CREBL2 may require specific lysis conditions to efficiently extract nuclear proteins.

How can I overcome challenges in detecting protein-protein interactions involving CREBL2?

To successfully detect CREBL2 protein-protein interactions:

• Crosslinking approach: Consider using membrane-permeable crosslinkers (DSP or formaldehyde) to stabilize transient interactions before cell lysis.

• Lysis buffer optimization: Use buffers containing 0.1-0.5% NP-40 or Triton X-100 with 150-300 mM NaCl to maintain interactions while ensuring efficient lysis.

• Co-immunoprecipitation strategies:

  • Tag-based approaches: Studies have successfully used myc-tagged CREBL2 to co-immunoprecipitate HA-tagged Crebrf .

  • Consider reverse co-IP to confirm interactions

  • Include appropriate negative controls (IgG, unrelated tagged protein)

• Alternative approaches:

  • Proximity ligation assay (PLA) for in situ detection of protein interactions

  • Split-reporter systems (BiFC, BRET, FRET) for monitoring interactions in living cells

  • Mass spectrometry following immunoprecipitation to identify novel interaction partners

• Interaction validation: Test interaction under different conditions, such as rapamycin treatment, which did not affect the amount of Crebrf co-immunoprecipitating with CREBL2 despite affecting downstream gene expression .

These approaches have successfully identified the CREBL2-Crebrf interaction, which appears to be conserved between Drosophila and mammalian systems.

How should I interpret changes in cellular triglyceride levels following CREBL2 manipulation?

When interpreting changes in cellular triglyceride (TAG) levels following CREBL2 manipulation:

• Baseline context: CREBL2 knockdown consistently increases TAG content in both C2C12 myoblasts and Hepa1-6 cells , indicating a conserved role in lipid metabolism regulation across cell types.

• Mechanistic considerations:

  • Increased TAG could result from enhanced fatty acid uptake from medium

  • Alternatively, cells might synthesize more fat de novo

  • Both C2C12 and Hepa1-6 cells with CREBL2 knockdown still increase TAG content when cultured in delipidated serum, suggesting enhanced de novo lipogenesis rather than increased uptake

• Transcriptional impact analysis:

  • Examine expression changes in genes involved in lipid metabolism

  • No single target gene knockdown recapitulates the full CREBL2 knockdown phenotype, suggesting combined reduction in expression of several genes may be responsible

• Integration with metabolic parameters:

  • Correlate with changes in glucose uptake and glycolysis (both elevated in CREBL2 knockdown)

  • Consider how changes relate to mTORC1 activity status

These interpretations should recognize that CREBL2's metabolic effects likely represent coordination of multiple cellular pathways rather than regulation of a single target gene.

What is the significance of CREBL2's role in the transcriptional response to mTORC1 inhibition?

The significance of CREBL2's role in mediating transcriptional responses to mTORC1 inhibition includes:

• Molecular integration: CREBL2 serves as a crucial link between nutrient sensing via mTORC1 and transcriptional reprogramming necessary for metabolic adaptation .

• Therapeutic implications: Understanding CREBL2's function may inform strategies to modulate mTORC1 pathway activity in diseases such as cancer, diabetes, and obesity, where metabolic dysregulation is central.

• Specificity of response:

  • CREBL2 is required for the appropriate activation of the majority of genes induced by rapamycin treatment

  • Some targets (e.g., Bloc1s1, Fbxo36) show no baseline change with CREBL2 knockdown but lose rapamycin responsiveness

  • Other targets (e.g., Cxcl12, Ing4) show elevated baseline expression with CREBL2 knockdown

• Conservation across species: The function of CREBL2 as a mediator of transcriptional changes downstream of mTORC1 appears conserved from Drosophila to mammals, highlighting its fundamental importance in cellular metabolism .

• Pathway integration: CREBL2 may coordinate multiple metabolic pathways, explaining why knockdown results in broad metabolic changes affecting glucose metabolism, glycolysis, and triglyceride biosynthesis .

This research positions CREBL2 as a key transcriptional regulator linking nutrient sensing to metabolic adaptation through the mTORC1 pathway.

How can I correlate CREBL2 expression data with functional metabolic outcomes?

To effectively correlate CREBL2 expression with functional metabolic outcomes:

• Establish a quantitative framework:

  • Measure CREBL2 expression across a range (partial knockdown to overexpression)

  • Quantify multiple metabolic parameters for each expression level

• Core metabolic measurements to include:

  • Triglyceride content (significantly increased with CREBL2 knockdown)

  • Glucose uptake (elevated in CREBL2 knockdown cells)

  • Glycolytic rate (assessed via lactate secretion, elevated with CREBL2 knockdown)

  • Mitochondrial respiration (oxygen consumption rate)

  • Fatty acid oxidation capacity

• Transcriptional correlation:

  • Perform RNA-Seq across CREBL2 expression levels

  • Identify genes with expression patterns that correlate with metabolic outcomes

  • Current data shows differences in gene expression patterns between C2C12 cells (408 genes downregulated, 164 upregulated) and Hepa1-6 cells (267 genes downregulated, 396 upregulated)

• Pathway response assessment:

  • Test how CREBL2 expression affects metabolic response to stimuli such as insulin, glucagon, or rapamycin

  • Determine whether CREBL2 is required for specific metabolic adaptations

• Mathematical modeling:

  • Develop predictive models of how CREBL2 expression levels influence metabolic outcomes

  • Validate models with experimental data from different cell types

This comprehensive approach will establish causative relationships between CREBL2 expression and metabolic phenotypes across contexts.

What are promising strategies for developing more specific CREBL2 antibodies for research applications?

Developing more specific CREBL2 antibodies remains challenging, as even experienced researchers have failed to generate specific antibodies against either CREBL2 or Crebrf . Promising strategies include:

• Epitope selection refinement:

  • Target highly unique regions of CREBL2 with low homology to other bZIP domain proteins

  • Consider using structural biology approaches to identify surface-exposed regions

  • Avoid the conserved bZIP domain that shares 41% identity with CREB

• Advanced antibody engineering:

  • Develop recombinant antibodies with targeted mutations to improve specificity

  • Apply CRISPR/HDR approaches for functional diversification of hybridoma-produced antibodies

  • Consider nanobody or single-chain variable fragment (scFv) formats that may access epitopes differently

• Validation improvements:

  • Implement parallel knockout/knockdown validation in multiple cell types

  • Develop comprehensive cross-reactivity panels with related proteins

  • Apply advanced imaging techniques like super-resolution microscopy for specificity assessment

• Alternative approaches when antibodies fail:

  • Tagged CREBL2 expression systems

  • CRISPR knock-in of small epitope tags into endogenous CREBL2

  • Proximity labeling approaches (BioID, APEX) to identify interacting partners without relying on direct CREBL2 antibodies

These strategies acknowledge past difficulties while leveraging newer technologies to overcome historical challenges in CREBL2 antibody development.

How might tissue-specific functions of CREBL2 inform therapeutic strategies?

Understanding tissue-specific CREBL2 functions could inform novel therapeutic approaches:

• Metabolic disease applications:

  • CREBL2's role in regulating triglyceride levels and glucose metabolism in muscle and liver cells suggests potential relevance for conditions like non-alcoholic fatty liver disease, insulin resistance, and type 2 diabetes

  • Tissue-specific modulation of CREBL2 activity could potentially address metabolic disorders without systemic effects

• Cancer therapeutic relevance:

  • Given CREBL2's location in a commonly deleted region in various cancers , understanding whether its loss is a driver or passenger event in specific tissues is crucial

  • Metabolic vulnerabilities created by CREBL2 loss might be exploitable in precision oncology approaches

• Research approaches for tissue specificity:

  • Conditional knockout models in specific tissues (liver, muscle, adipose)

  • Single-cell transcriptomics to identify cell type-specific CREBL2 regulatory networks

  • In vivo metabolic phenotyping of tissue-specific CREBL2 manipulation

• Therapeutic development considerations:

  • Small molecules targeting the CREBL2-Crebrf interaction

  • Tissue-specific delivery systems for CREBL2-modulating agents

  • Metabolic pathway interventions that compensate for CREBL2 dysfunction

This tissue-specific understanding will be essential for translating basic CREBL2 biology into therapeutic applications with minimal off-target effects.

What is the relationship between CREBL2 and diseases associated with metabolic dysregulation?

The relationship between CREBL2 and metabolic dysregulation diseases warrants further investigation:

• Potential disease associations:

  • Non-alcoholic fatty liver disease: CREBL2 knockdown increases triglyceride content in hepatic cells

  • Insulin resistance and diabetes: CREBL2 regulates glucose uptake and glycolysis

  • Cancer metabolism: CREBL2 is in a chromosomal region frequently deleted in various cancers

  • Adipose tissue disorders: CREBL2 is involved in adipocyte differentiation

• Molecular mechanisms to investigate:

  • How CREBL2 expression correlates with disease progression

  • Whether CREBL2 genetic variants are associated with metabolic disease risk

  • How CREBL2's interaction with mTORC1 signaling influences disease states

• Research approaches:

  • Analysis of CREBL2 expression in patient samples across metabolic disease states

  • Metabolic phenotyping of disease models with CREBL2 manipulation

  • Pharmacological studies using mTORC1 inhibitors in the context of altered CREBL2 expression

• Translational implications:

  • Potential biomarker development based on CREBL2 expression or activity

  • Therapeutic targeting of pathways downstream of CREBL2

  • Personalized medicine approaches for patients with altered CREBL2 expression

This research direction could establish CREBL2 as a key molecular link between mTORC1 signaling and metabolic diseases, potentially identifying new therapeutic targets or biomarkers.