MEF2C Antibody

What biological functions does MEF2C regulate?

MEF2C functions as a transcription activator with roles spanning multiple physiological systems. It controls cardiac morphogenesis and myogenesis, and is involved in vascular development. In the nervous system, MEF2C plays an essential role in hippocampal-dependent learning and memory by suppressing the number of excitatory synapses and regulating basal and evoked synaptic transmission . It is crucial for normal neuronal development, distribution, and electrical activity in the neocortex.

Beyond neurodevelopment, MEF2C is necessary for proper development of megakaryocytes, platelets, and bone marrow B-lymphopoiesis. It is required for B-cell survival and proliferation in response to BCR stimulation, efficient IgG1 antibody responses to T-cell-dependent antigens, and normal induction of germinal center B-cells .

What are the typical applications for MEF2C antibodies?

MEF2C antibodies are utilized across numerous experimental applications. Based on validated research protocols, the primary applications include:

These applications have been validated across multiple research publications, with Western blot being the most commonly reported technique (26 publications), followed by immunofluorescence (16 publications), and immunohistochemistry (10 publications) .

What is the expected molecular weight for MEF2C in Western blot analysis?

While the calculated molecular weight of MEF2C is approximately 51 kDa, the observed molecular weight in Western blot analyses typically ranges from 45-70 kDa . This variation may result from:

• Post-translational modifications (phosphorylation, SUMOylation)

• Different isoforms of MEF2C

• Tissue-specific expression patterns

For instance, in detection of human MEF2C by Western blot using K562 human chronic myelogenous leukemia cell line lysates, specific bands were detected at approximately 52-60 kDa . It's advisable to include positive control samples (such as SH-SY5Y cells, mouse brain tissue, or mouse heart tissue) when establishing a new Western blot protocol for MEF2C detection .

What cell and tissue types are optimal for MEF2C detection?

MEF2C expression has been well-documented in specific cell and tissue types, making them preferable for antibody validation and experimental controls:

MEF2C displays primarily nuclear localization, consistent with its function as a transcription factor. In the brain, MEF2C staining is localized specifically to neuronal nuclei, as demonstrated in both human hippocampus and mouse cortex samples .

How can specificity of MEF2C antibodies be validated?

Validating antibody specificity is crucial for reliable research outcomes. For MEF2C antibodies, multiple validation approaches are recommended:

• Knockout/Knockdown Verification: Use of MEF2C knockout cell lines, such as MEF2C knockout THP-1 cells, provides the most definitive validation. Western blot analysis should show absence of bands in knockout samples compared to wild-type controls .

• Peptide Competition Assays: Pre-incubation of the antibody with immunizing peptide should abolish specific signal in applications like Western blot and immunohistochemistry.

• Electrophoretic Mobility Shift Assay (EMSA): For antibodies intended for supershift assays, validation can be performed using wild-type MEF2 oligonucleotides (5′-GCACTTGGCT-TATTTTTAGAAAGACT) versus mutant oligonucleotides (5′-GCACTTGGCTTGTTCTTAGAAAGACT) .

• Multiple Antibody Comparison: Using different antibodies targeting distinct epitopes of MEF2C should yield similar pattern of expression in the same experimental system.

• Subcellular Localization: MEF2C should primarily localize to the nucleus, consistent with its function as a transcription factor.

What are the optimal protocols for studying MEF2C in neuronal development?

MEF2C plays critical roles in neuronal development, with the following recommended protocols for its investigation:

• Immunohistochemistry of Brain Sections:

  • For paraffin-embedded sections: Use antigen retrieval with TE buffer pH 9.0 or citrate buffer pH 6.0

  • For frozen sections: Fix with 4% PFA prior to antibody incubation

  • Optimal antibody concentration: 1-5 μg/mL with overnight incubation at 4°C

  • Detection: Anti-species HRP-DAB staining kit with hematoxylin counterstain

  • Expected result: Nuclear staining in neuronal populations

• Cultured Neuron Analysis:

  • MEF2C regulates excitatory and inhibitory synaptic development

  • Combined immunofluorescence for MEF2C with synaptic markers (PSD-95, gephyrin)

  • Dendritic spine analysis using MEF2C conditional knockout models reveals decreased spine density

• Neuronal Network Activity:

  • MEF2C loss affects UP states in cortical neurons

  • Electrophysiological recording of layer 2/3 pyramidal neurons shows decreased excitatory and increased inhibitory synaptic transmission upon MEF2C deletion

How does post-transcriptional regulation affect MEF2C expression?

Recent research has revealed important insights into MEF2C post-transcriptional regulation:

• m6A RNA Modification: MEF2C mRNA is subjected to N6-methyladenosine (m6A) modification, which regulates its expression during myogenic differentiation. This modification is mediated by the methyltransferase METTL3 .

• Reader Protein Involvement: YTHDF1, an m6A reader protein, directly binds to MEF2C mRNA by recognizing the m6A modification site, promoting MEF2C expression at the translational level .

• Experimental Evidence:

  • Dual-luciferase reporter assays with wild-type (MEF2C-WT) and mutated (MEF2C-MUT) reporters show differential activity in METTL3 knockdown or overexpression conditions

  • RNA immunoprecipitation (RIP-qPCR) confirms YTHDF1 binding to MEF2C transcripts

  • Mutation of m6A sites in MEF2C mRNA reduces protein expression and affects myotube formation

• Implications for Antibody Detection: These post-transcriptional modifications may contribute to variations in MEF2C protein levels detected by antibodies, potentially explaining discrepancies between mRNA and protein abundance in certain experimental systems.

How can I optimize ChIP experiments using MEF2C antibodies?

Chromatin immunoprecipitation (ChIP) with MEF2C antibodies requires specific optimization steps:

• Cell Preparation Protocol:

  • Fixation: Treat cells with formaldehyde (1% final concentration) for 10 minutes at room temperature

  • Chromatin preparation: Follow standard X-ChIP protocol

  • Recommended chromatin amount: 25 μg per immunoprecipitation

• Antibody Selection and Amount:

  • Use ChIP-validated antibodies (e.g., ab211493)

  • Optimal antibody amount: 5 μg per 25 μg of chromatin

  • Include proper controls: Normal IgG (5 μg) and protein A/G sepharose beads (20 μl slurry)

• Target Validation:

  • Quantify immunoprecipitated DNA by real-time PCR using SYBR green chemistry

  • Analyze enrichment at known MEF2C binding sites

  • Expected results: Significant enrichment at MEF2 consensus sites compared to IgG control

• Specific Considerations:

  • MEF2C binding motif consensus sequence: 5′-CTAAAAATAG-3′ and variants

  • Prepare nuclear extracts for optimal chromatin isolation

  • Use sonication to generate 200-500 bp chromatin fragments

What is the relationship between MEF2C and B-cell development?

MEF2C plays critical roles in B-cell development and function:

• B-cell Development Markers:

  • MEF2C expression can be monitored across B-cell development stages using B220, CD43, BP-1, CD24, IgM, and IgD markers

  • In conditional knockout models (Mef2c-cKO), B-cell populations can be analyzed by flow cytometry

• Functional Impact:

  • MEF2C is required for B-cell survival and proliferation in response to BCR stimulation

  • While BCR-induced calcium mobilization remains intact after Mef2c deletion, B-cell proliferation and survival are impaired

  • Required for efficient IgG1 antibody responses to T-cell-dependent antigens

  • Necessary for normal induction of germinal center B-cells

• Experimental Approaches:

  • Use of conditional knockout models (e.g., Mef2c fl/fl Cd19+/cre) provides insights into B-cell-specific functions

  • Flow cytometry analysis of B-cell populations using established markers

  • Proliferation assays following BCR stimulation

  • Analysis of antibody responses to T-cell-dependent antigens

How can I optimize Western blot protocols for MEF2C detection?

Western blot optimization for MEF2C requires attention to several technical aspects:

• Sample Preparation:

  • Nuclear extraction is critical as MEF2C is primarily nuclear

  • Recommended protocol: Use NE-PER nuclear and cytoplasmic extraction reagents with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is relevant

• Gel Percentage and Transfer Conditions:

  • Use 8-10% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membrane is recommended over nitrocellulose

  • Transfer buffer containing 20% methanol at 100V for 1 hour or 30V overnight at 4°C

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:1000

  • Incubation: Overnight at 4°C in 5% non-fat milk or BSA in TBS-T

  • Secondary antibody: Anti-species HRP conjugates at 1:10,000-1:20,000

  • For fluorescent detection: Secondary antibodies such as Goat anti-Rabbit IgG H&L 800CW at 1:20,000 dilution

• Controls and Validation:

  • Positive controls: SH-SY5Y cells, mouse brain tissue, mouse heart tissue

  • Negative controls: MEF2C knockout cell lines when available

  • Expected band size: 45-70 kDa range, with most common observation at 52-60 kDa

What factors affect variability in MEF2C immunohistochemistry results?

Several factors can influence the consistency and specificity of MEF2C detection in IHC:

• Fixation and Antigen Retrieval:

  • Formaldehyde fixation can mask epitopes

  • Recommended antigen retrieval: TE buffer pH 9.0 or citrate buffer pH 6.0

  • Duration of antigen retrieval: 15-20 minutes at 95-100°C

• Tissue-Specific Considerations:

  • Brain tissue: Perfusion fixation provides better results than immersion fixation

  • Muscle tissue: May require shorter fixation times to prevent over-fixation

  • Human tissues: Postmortem interval affects antibody performance

• Antibody Selection and Protocol:

  • Polyclonal antibodies may show batch-to-batch variability

  • Monoclonal antibodies provide more consistent results but may recognize fewer epitopes

  • Optimal antibody concentration: Typically 1-5 μg/mL for IHC

  • Incubation conditions: Overnight at 4°C provides optimal signal-to-noise ratio

• Detection Systems:

  • HRP-DAB systems work well for MEF2C detection in most tissues

  • When examining co-localization, fluorescent secondary antibodies may be preferable

  • Anti-Sheep HRP-DAB Cell & Tissue Staining Kit has been validated for MEF2C detection

How do I interpret MEF2C expression changes in different experimental contexts?

Interpreting MEF2C expression changes requires consideration of multiple factors:

• Developmental Context:

  • MEF2C expression changes throughout development

  • In neurons, MEF2C regulates excitatory/inhibitory synapse balance

  • In muscle, MEF2C expression increases during myogenic differentiation

• Cellular Localization:

  • Primary localization: Nuclear

  • Changes in nuclear vs. cytoplasmic distribution may indicate altered activity

  • Phosphorylation can affect subcellular localization

• Isoform Considerations:

  • Multiple MEF2C isoforms exist

  • Isoforms lacking the repressor domain are more active than isoform 1

  • Different antibodies may have varying affinities for specific isoforms

• Quantification Approaches:

  • Western blot: Normalize to nuclear markers (e.g., Lamin B) rather than cytoplasmic proteins

  • Immunofluorescence: Measure nuclear intensity relative to background

  • Consider relative vs. absolute quantification methods depending on experimental question

How can MEF2C antibodies be used to study neurodevelopmental disorders?

MEF2C is implicated in several neurodevelopmental disorders, making it an important research target:

• Autism Spectrum Disorders (ASD) and Intellectual Disability (ID):

  • MEF2C loss-of-function produces mice with behavior phenotypes reminiscent of ASD and ID

  • Behavioral changes associated with altered E/I synapse densities and reduced cortical network activity

  • MEF2C regulates numerous autism- and synapse-linked genes

• Research Approaches:

  • Conditional knockout models (e.g., Emx1-Cre;Mef2c-flox) allow cell type-specific analysis

  • Electrophysiological recordings combined with immunohistochemistry

  • Single-cell transcriptomics to identify MEF2C-regulated gene networks

• Translational Applications:

  • Analysis of MEF2C expression in patient-derived samples

  • Correlation of MEF2C levels with clinical phenotypes

  • Use of MEF2C antibodies in screening potential therapeutic compounds

What are the latest techniques for studying MEF2C-DNA interactions?

Advanced methods for studying MEF2C-DNA interactions include:

• ChIP-seq:

  • Genome-wide profiling of MEF2C binding sites

  • Requires ChIP-grade antibodies with high specificity

  • Analysis reveals MEF2C binding at regulatory regions of muscle-specific genes

• CUT&RUN/CUT&Tag:

  • Higher signal-to-noise ratio than traditional ChIP

  • Requires fewer cells

  • Compatible with MEF2C antibodies validated for immunoprecipitation

• HiChIP/PLAC-seq:

  • Combines chromatin conformation capture with ChIP

  • Identifies long-range interactions mediated by MEF2C

  • Requires optimization of cross-linking and antibody conditions

• CRISPR/Cas9-based approaches:

  • CUT&Tag combined with targeted degradation of MEF2C

  • Rapid analysis of MEF2C-dependent chromatin modifications

  • Generation of tagged endogenous MEF2C for antibody-independent analysis

How is MEF2C regulated by post-translational modifications, and how can these be studied?

MEF2C undergoes various post-translational modifications that regulate its activity:

• Phosphorylation:

  • Multiple kinases phosphorylate MEF2C at different residues

  • Phospho-specific antibodies can track modification status

  • Phosphorylation can either enhance or inhibit transcriptional activity

• SUMOylation and Ubiquitination:

  • Affect MEF2C stability and activity

  • Can be studied using immunoprecipitation followed by Western blot with modification-specific antibodies

  • SUMO-specific proteases regulate MEF2C activity

• Acetylation:

  • Histone deacetylases (HDACs) interact with MEF2C

  • Acetylation status affects DNA binding activity

  • Can be analyzed using acetylation-specific antibodies

• Experimental Approaches:

  • Mass spectrometry to identify novel modifications

  • Site-directed mutagenesis of modification sites

  • Pharmacological manipulation of modifying enzymes

  • Immunoprecipitation with modification-specific antibodies

What are the best practices for selecting MEF2C antibodies for specific applications?

When selecting MEF2C antibodies, researchers should consider:

• Application Compatibility:

  • Verify antibody validation for your specific application (WB, IHC, ChIP, etc.)

  • Review published literature using the antibody for similar applications

  • Check reactivity with species of interest (human, mouse, rat, etc.)

• Epitope Considerations:

  • Antibodies targeting different regions may perform differently

  • N-terminal antibodies may detect more isoforms

  • C-terminal antibodies may miss truncated variants

• Validation Status:

  • Knockout/knockdown validation provides highest confidence

  • Multiple antibody comparison increases reliability

  • Check for cross-reactivity with other MEF2 family members

• Technical Support:

  • Available protocols specific to your application

  • Published validation data

  • Citations in peer-reviewed literature

What emerging research areas involve MEF2C function?

MEF2C research is expanding into several emerging areas:

• Single-Cell Transcriptomics:

  • Cell type-specific expression patterns of MEF2C

  • Identification of MEF2C-regulated gene networks at single-cell resolution

  • Temporal dynamics of MEF2C expression during development

• Neuropsychiatric Disorders:

  • MEF2C mutations in autism, intellectual disability, and schizophrenia

  • Role in excitatory/inhibitory balance in neuronal circuits

  • Potential therapeutic target for neurodevelopmental disorders

• Immune System Function:

  • B-cell development and function

  • Response to infection and vaccination

  • Potential role in autoimmune disorders

• Regenerative Medicine:

  • Role in muscle regeneration and cardiac repair

  • Potential target for enhancing tissue regeneration

  • Function in stem cell differentiation pathways