JMJD1C Antibody

What is JMJD1C and why is it important in research?

JMJD1C is a histone demethylase that primarily targets H3K9 methylation marks. It functions as a critical epigenetic regulator involved in transcriptional activation. JMJD1C has been identified as an essential coactivator for RUNX1-RUNX1T1 (formerly AML1-ETO) in acute myeloid leukemia (AML), where it dictates leukemic programs by increasing self-renewal and inhibiting differentiation . Additionally, JMJD1C plays important roles in cardiac hypertrophy through regulation of the CAMKK2-AMPK signaling pathway . The diverse functions of JMJD1C in different cellular contexts make it an important target for research in cancer biology, cardiovascular disease, and epigenetic regulation.

What types of JMJD1C antibodies are available for research applications?

Several types of JMJD1C antibodies are available for research applications, varying in their binding specificity, host organism, clonality, and conjugation status. The available antibodies include:

• Monoclonal antibodies targeting specific epitopes, such as those recognizing amino acids 2-99 of JMJD1C (e.g., clone 5F12)

• Polyclonal antibodies targeting different regions, including N-terminal, C-terminal, and internal domains

• Antibodies with various conjugates (unconjugated, HRP, FITC, Biotin) for different detection methods

• Antibodies raised in different host organisms, predominantly mouse and rabbit

The selection of an appropriate antibody depends on the specific research application, required sensitivity, and experimental conditions.

What are the recommended applications for JMJD1C antibodies?

JMJD1C antibodies can be utilized in various experimental applications including:

• Western blotting (WB) for protein expression analysis

• Immunohistochemistry (IHC) on paraffin-embedded sections to visualize protein localization in tissues

• Enzyme-linked immunosorbent assay (ELISA) for quantitative protein detection

• Immunoprecipitation (IP) for protein-protein interaction studies

• Chromatin immunoprecipitation (ChIP) to identify genomic binding sites

For instance, custom-made rabbit polyclonal JMJD1C antibodies have been successfully used in both immunoprecipitation and immunoblotting experiments to specifically recognize JMJD1C, but not its paralogs JMJD1A and JMJD1B . Similarly, JMJD1C antibodies have been employed in ChIP assays to determine binding to the promoters of genes like CAMKK2, FHL1, and β-MHC .

How should I validate a JMJD1C antibody for my specific application?

Validating a JMJD1C antibody for your specific application is crucial to ensure reliable results. A comprehensive validation approach should include:

• Specificity testing: Compare signal in cells with endogenous JMJD1C expression versus cells with JMJD1C knockdown or knockout. Custom antibodies have been validated to specifically recognize JMJD1C without cross-reactivity to paralogs JMJD1A and JMJD1B .

• Positive and negative controls: Include known positive samples (tissues or cell lines with confirmed JMJD1C expression) and negative controls (tissues or cell lines with absent or reduced JMJD1C expression).

• Cross-reactivity assessment: Test the antibody against recombinant JMJD1C protein and assess potential cross-reactivity with related proteins, particularly JMJD1A and JMJD1B.

• Application-specific validation: For ChIP applications, perform sequential ChIP with different antibodies targeting the same complex. For co-immunoprecipitation studies, verify using reciprocal immunoprecipitations as demonstrated in Kasumi-1 cells .

What are the optimal conditions for using JMJD1C antibodies in Western blotting?

When performing Western blotting with JMJD1C antibodies, consider the following optimization steps:

• Sample preparation: JMJD1C is a large protein (~310 kDa), requiring careful sample preparation to prevent degradation. Use fresh samples with protease inhibitors.

• Gel selection: Use low percentage gels (4-6%) or gradient gels to resolve this high molecular weight protein effectively.

• Transfer conditions: Optimize transfer conditions for large proteins, potentially using longer transfer times or wet transfer systems rather than semi-dry methods.

• Blocking conditions: Test different blocking agents (BSA vs. non-fat dry milk) to reduce background while maintaining specific signal.

• Antibody dilution: Start with the manufacturer's recommended dilution and optimize as needed. For monoclonal antibodies like clone 5F12, which targets amino acids 2-99 of JMJD1C, follow specific recommendations for that clone .

• Detection method: Consider enhanced chemiluminescence for standard detection or fluorescent secondary antibodies for quantitative analysis.

How can I optimize JMJD1C antibodies for chromatin immunoprecipitation (ChIP) experiments?

For optimal results in ChIP experiments with JMJD1C antibodies:

• Crosslinking optimization: Test different formaldehyde concentrations (0.5-1%) and incubation times to achieve optimal crosslinking without overfix.

• Sonication conditions: Carefully optimize sonication to achieve chromatin fragments of 200-500 bp without damaging the epitopes recognized by the antibody.

• Antibody selection: Choose antibodies specifically validated for ChIP applications. Custom-made rabbit polyclonal JMJD1C antibodies have been successfully used in ChIP-seq analyses in Kasumi-1 cells .

• Controls: Include appropriate controls such as IgG negative controls and positive controls targeting known abundant histone marks.

• Enrichment analysis: Normalize your ChIP data to input samples and analyze enrichment at known JMJD1C binding sites. JMJD1C has been shown to bind to promoters of genes like CAMKK2, which can serve as positive controls .

• Sequential ChIP: Consider sequential ChIP (ChIP-reChIP) if studying JMJD1C in complexes with other proteins, such as RUNX1-RUNX1T1 in AML cells .

How does JMJD1C interact with transcriptional complexes in AML cells?

JMJD1C plays a critical role in acute myeloid leukemia (AML) through its interaction with transcriptional complexes:

• JMJD1C physically associates with RUNX1-RUNX1T1 (AML1-ETO) in Kasumi-1 cells, as demonstrated by reciprocal coimmunoprecipitation experiments .

• The interaction between JMJD1C and RUNX1-RUNX1T1 is direct and stable, as shown using Flag-tagged JMJD1C and HA-tagged RUNX1-RUNX1T1 proteins expressed in and purified from insect cells .

• JMJD1C interacts not only with RUNX1-RUNX1T1 but also with all components of the AETFC (AML1-ETO transcription factor complex), including HEB, as demonstrated by coimmunoprecipitation experiments .

• The interaction between JMJD1C and RUNX1-RUNX1T1 is stronger in the context of the complete AETFC complex compared to RUNX1-RUNX1T1 alone, suggesting cooperative binding involving multiple complex components .

• JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program, maintaining low H3K9 dimethyl (H3K9me2) levels at target genes .

What role does JMJD1C play in cardiac hypertrophy?

JMJD1C has been identified as a regulator of cardiac hypertrophy through several mechanisms:

• JMJD1C is overexpressed in hypertrophic hearts of humans and mice, correlating with reduced histone methylation levels .

• In cardiomyocytes, JMJD1C knockdown represses angiotensin II (Ang II)-mediated increases in cell size and expression of hypertrophic genes, while JMJD1C overexpression promotes hypertrophic responses .

• Mechanistically, JMJD1C regulates AMP-dependent kinase (AMPK) activation in cardiomyocytes by repressing CAMKK2 expression rather than influencing LKB1 .

• JMJD1C directly binds to the CAMKK2 promoter, as demonstrated by chromatin immunoprecipitation assays, and JMJD1C overexpression reduces H3K9me1 enrichment at this site .

• The effects of JMJD1C on AMPK activation can be blocked by the CAMKK2 inhibitor STO609, confirming the CAMKK2-dependent mechanism .

• AMPK knockdown blocks the inhibitory functions of JMJD1C knockdown on Ang II-induced hypertrophic responses, while the AMPK activator metformin reduces the pro-hypertrophic functions of JMJD1C in cardiomyocytes .

How can I assess JMJD1C's demethylase activity in experimental systems?

Assessing JMJD1C's histone demethylase activity requires multiple approaches:

• Immunoblotting for histone modifications: Examine global levels of H3K9me1, H3K9me2, and H3K9me3 modifications after JMJD1C knockdown or overexpression. Studies have shown correlations between JMJD1C expression and decreased H3K9 methylation marks in cardiac hypertrophy models .

• ChIP assays: Perform ChIP for H3K9 methylation marks at specific loci known to be regulated by JMJD1C. For instance, JMJD1C overexpression reduces H3K9me1 enrichment at the CAMKK2 promoter in cardiomyocytes .

• In vitro demethylase assays: Use recombinant JMJD1C protein with synthetic histone peptides containing specific methylation marks to directly measure enzymatic activity.

• Mass spectrometry: Employ quantitative mass spectrometry to analyze changes in histone modifications upon JMJD1C manipulation.

• Gene expression correlation: Analyze correlations between JMJD1C binding, H3K9 methylation status, and gene expression changes using integrated genomic approaches combining ChIP-seq and RNA-seq data.

What are common issues when using JMJD1C antibodies and how can I resolve them?

When working with JMJD1C antibodies, researchers often encounter several challenges:

• High molecular weight detection issues: JMJD1C is a large protein (~310 kDa), which can make detection challenging. Ensure complete transfer from gel to membrane by using appropriate transfer conditions for high molecular weight proteins. Consider using gradient gels and wet transfer systems with extended transfer times.

• Specificity concerns: Some antibodies may cross-react with JMJD1A or JMJD1B. Use antibodies specifically validated against these paralogs, like the custom rabbit polyclonal antibody described in the literature . Always include appropriate positive and negative controls.

• Batch-to-batch variability: Different lots of the same antibody may perform differently. Maintain consistent antibody lots for long-term projects or revalidate new lots against previous ones.

• Signal-to-noise issues: Optimize blocking conditions, antibody dilutions, and washing steps. Consider using more sensitive detection methods for low-abundance applications.

• Fixation-sensitive epitopes: For IHC applications, test different fixation protocols as some epitopes may be sensitive to certain fixatives or antigen retrieval methods.

How can I differentiate between JMJD1C and its paralogs (JMJD1A, JMJD1B) in my experiments?

Distinguishing JMJD1C from its paralogs JMJD1A and JMJD1B requires careful experimental design:

• Antibody selection: Use antibodies specifically validated to recognize JMJD1C without cross-reactivity to JMJD1A or JMJD1B. Custom-made antibodies have been developed that specifically recognize JMJD1C in both immunoprecipitation and immunoblotting experiments .

• Molecular weight discrimination: JMJD1C (~310 kDa) is larger than JMJD1A and JMJD1B, which can help distinguish these proteins in Western blotting applications.

• Isoform-specific primers: Design PCR primers targeting unique regions of each paralog for mRNA expression analysis.

• Functional validation: Perform targeted knockdown experiments using specific siRNAs or shRNAs against each paralog. Studies have shown that depletion of JMJD1A or JMJD1B does not affect RUNX1-RUNX1T1's ability to inhibit differentiation, while JMJD1C depletion does, demonstrating functional specificity .

• Domain-specific analysis: Focus on regions of JMJD1C that differ from its paralogs, such as using antibodies targeting amino acids 2-99, which may contain sequences unique to JMJD1C .

What controls should I include when using JMJD1C antibodies in ChIP experiments?

For robust ChIP experiments with JMJD1C antibodies, include these essential controls:

• IgG negative control: Always include matched IgG (same species and isotype as your JMJD1C antibody) as a negative control to establish background signal levels .

• Input DNA control: Save a portion of sonicated chromatin before immunoprecipitation to normalize ChIP signals and account for differences in starting material.

• Positive control regions: Include PCR primers for regions known to be bound by JMJD1C, such as the CAMKK2 promoter in cardiomyocytes .

• Negative control regions: Include primers for genomic regions not expected to bind JMJD1C.

• Knockdown/knockout validation: Perform ChIP in cells with JMJD1C knockdown or knockout to confirm antibody specificity.

• Sequential ChIP: For complex interactions, consider sequential ChIP to verify co-occupancy with known JMJD1C binding partners such as RUNX1-RUNX1T1 in AML cells .

• Spike-in controls: Consider using exogenous spike-in chromatin from another species with a species-specific antibody to control for technical variation between samples.

How can JMJD1C antibodies be used to investigate protein-protein interactions in transcriptional complexes?

JMJD1C antibodies are valuable tools for investigating protein-protein interactions within transcriptional complexes:

• Coimmunoprecipitation (Co-IP): JMJD1C antibodies can be used to pull down JMJD1C and its associated proteins. Reciprocal Co-IP experiments with JMJD1C and RUNX1-RUNX1T1 antibodies have demonstrated their physical association in Kasumi-1 cells .

• Mass spectrometry-based approaches: Following immunoprecipitation with JMJD1C antibodies, mass spectrometry can identify novel interaction partners in an unbiased manner.

• Proximity ligation assays (PLA): This technique can visualize protein-protein interactions in situ by detecting proteins in close proximity using specific antibodies and rolling circle amplification.

• Immunofluorescence colocalization: Colocalization of JMJD1C with potential interaction partners can be assessed using fluorescently labeled antibodies and confocal microscopy.

• ChIP-reChIP: Sequential ChIP with JMJD1C antibodies followed by ChIP with antibodies against potential interaction partners can identify co-occupancy at specific genomic loci.

• FRET/BRET analysis: These techniques can assess direct protein-protein interactions using appropriately labeled antibodies or fusion proteins.

What strategies can be employed to study JMJD1C function in leukemia and cardiac disease models?

To investigate JMJD1C function in disease models, researchers can employ several strategies:

In leukemia models:

• Genetic manipulation: Knockdown or knockout JMJD1C in AML cell lines (e.g., Kasumi-1, SKNO-1) to assess effects on cell proliferation, differentiation, and survival .

• Colony formation assays: Evaluate the impact of JMJD1C depletion on colony-forming ability of AML cells or progenitor cells expressing RUNX1-RUNX1T1 .

• Differentiation assays: Assess how JMJD1C manipulation affects differentiation block in AML cells by measuring surface markers like CD11b following treatment with differentiation-inducing agents .

• Integrated genomic approaches: Combine ChIP-seq, RNA-seq, and ATAC-seq to comprehensively map JMJD1C binding sites, associated chromatin changes, and gene expression alterations.

In cardiac disease models:

• In vitro hypertrophy models: Manipulate JMJD1C expression in cardiomyocytes treated with hypertrophic stimuli like angiotensin II and assess cell size and hypertrophic gene expression .

• Signaling pathway analysis: Investigate how JMJD1C affects the CAMKK2-AMPK pathway by measuring phosphorylation status and expression levels of key components .

• In vivo models: While current studies are based on in vitro cardiomyocyte models, development of cardiomyocyte-specific JMJD1C knockout mouse lines would allow investigation of in vivo functions during cardiac hypertrophy .

• Pharmacological interventions: Test how JMJD1C function is affected by drugs targeting related pathways, such as metformin for AMPK activation or STO609 for CAMKK2 inhibition .

How can genome-wide approaches be combined with JMJD1C antibodies to understand global regulatory networks?

Integrating JMJD1C antibodies with genome-wide approaches provides powerful insights into global regulatory networks:

• ChIP-seq: Using JMJD1C antibodies for chromatin immunoprecipitation followed by high-throughput sequencing can identify genome-wide binding sites. This approach has been applied to map JMJD1C occupancy in Kasumi-1 cells and compare with RUNX1-RUNX1T1 binding sites .

• CUT&RUN or CUT&Tag: These newer techniques offer improved signal-to-noise ratios compared to traditional ChIP-seq and can be performed with fewer cells.

• ChIP-seq for histone modifications: Parallel ChIP-seq for H3K9 methylation marks can determine how JMJD1C binding correlates with changes in these specific modifications across the genome.

• Hi-ChIP or PLAC-seq: These methods combine chromatin immunoprecipitation with chromosome conformation capture to identify long-range interactions mediated by JMJD1C.

• Multi-omics integration: Correlate JMJD1C binding sites with transcriptomic (RNA-seq), chromatin accessibility (ATAC-seq), and other epigenomic data to construct comprehensive regulatory networks.

• Single-cell approaches: Emerging single-cell epigenomic methods could reveal cell-to-cell variability in JMJD1C binding and function within heterogeneous populations like tumor samples.