L3MBTL4 Antibody

What is L3MBTL4 and what is its biological significance?

L3MBTL4 is lethal(3) malignant brain tumor-like protein 4, a protein that functions primarily in the nucleus. Recent genome-wide association studies have identified L3MBTL4 as a novel susceptibility gene significantly associated with hypertension (meta-analyses odds ratio = 1.15, 95% confidence interval = 1.07–1.23, P = 6.128 × 10^-9) . Functionally, L3MBTL4 is predominantly expressed in vascular smooth muscle cells and has been found to be up-regulated in spontaneously hypertensive rats . The biological significance of L3MBTL4 lies in its ability to regulate vascular remodeling by down-regulating latent transforming growth factor-β binding protein 1 (LTBP1) and activating the mitogen-activated protein kinases (MAPK) signaling pathway, which triggers pathological progression of vascular remodeling and blood pressure elevation .

What applications are L3MBTL4 antibodies suitable for?

L3MBTL4 antibodies, such as the commercially available 26280-1-AP, are suitable for several research applications:

For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may be used alternatively . The antibody has been demonstrated to show reactivity with human and mouse samples, making it suitable for comparative studies across these species .

What is the cellular localization of L3MBTL4?

L3MBTL4 is primarily localized in the nucleus of cells, which is consistent with its presumed function in gene regulation. This nuclear localization has been experimentally verified in vascular smooth muscle cells (VSMCs) . In vessel tissue, immunofluorescence staining has shown that L3MBTL4 co-localizes with α-actin, indicating its predominant expression in the medial layer of the vasculature . Double immunofluorescence staining techniques are recommended when studying the co-localization of L3MBTL4 with other cellular markers, using the appropriate fixation methods to preserve nuclear architecture while maintaining antigen accessibility .

What is the observed molecular weight of L3MBTL4 in Western blot?

The observed molecular weight of L3MBTL4 in Western blot applications is 75-80 kDa . When performing Western blot analysis for L3MBTL4, researchers should prepare protein samples in standard reducing conditions and use 8-10% SDS-PAGE gels for optimal separation. For primary antibody incubation, dilutions of 1:500-1:1000 are typically effective, though optimization may be necessary depending on the specific experimental conditions and sample types . Positive controls, such as mouse testis tissue, where L3MBTL4 expression has been validated, are recommended when establishing Western blot protocols .

What tissues and cell types show high expression of L3MBTL4?

L3MBTL4 shows differential expression across tissues and cell types. According to the available research data:

• Blood vessels: L3MBTL4 is highly expressed in vascular tissue, particularly in spontaneously hypertensive rats (SHRs) compared to normotensive Wistar-Kyoto rats (WKYs) .

• Cellular expression: Among different human cell lines, L3MBTL4 is highly expressed in smooth muscle cells (SMCs) and endothelial cells .

• Subcellular distribution: Within the vascular system, L3MBTL4 is predominantly found in the medial layer of blood vessels, co-localizing with α-actin, which is a marker for smooth muscle cells .

• Other tissues: Positive Western blot detection has been reported in mouse testis tissue, suggesting expression in reproductive tissues as well .

For researchers studying L3MBTL4 expression patterns, quantitative PCR and Western blotting comparing different tissues from hypertensive and normotensive models are recommended methodological approaches .

How should I optimize immunohistochemistry protocols for L3MBTL4 detection in vascular tissue?

Optimizing immunohistochemistry (IHC) protocols for L3MBTL4 detection in vascular tissue requires careful attention to several key parameters:

• Tissue fixation: For vascular tissue, 4% paraformaldehyde fixation for 24-48 hours followed by paraffin embedding is recommended to preserve tissue architecture while maintaining antigen integrity.

• Antigen retrieval: Based on validated protocols, use TE buffer pH 9.0 for optimal antigen retrieval. Heat-induced epitope retrieval should be performed at 95-98°C for 15-20 minutes . As an alternative, citrate buffer pH 6.0 can be used if TE buffer yields suboptimal results.

• Antibody dilution: Start with a 1:100 dilution for vascular tissue sections and optimize as needed. The recommended range is 1:50-1:500 .

• Detection system: For vascular tissue, a polymer-based detection system often provides better signal-to-noise ratio than avidin-biotin systems due to endogenous biotin in vascular tissues.

• Counterstaining: Hematoxylin counterstaining should be brief (2-3 minutes) to avoid masking nuclear L3MBTL4 signal.

• Controls: Include both positive controls (such as sections from hypertensive rat aorta, which has been shown to express higher levels of L3MBTL4) and negative controls (primary antibody omission) .

• Co-staining: For co-localization studies, double immunofluorescence staining with α-actin antibodies helps to confirm L3MBTL4 expression in the vascular media layer .

What are the best experimental approaches to study L3MBTL4's role in the MAPK signaling pathway?

To study L3MBTL4's role in the MAPK signaling pathway, several complementary approaches can be employed:

• Phosphorylation analysis: Western blotting with phospho-specific antibodies against p38MAPK, JNK, and ERK to assess activation states in systems with varied L3MBTL4 expression. This should be performed in both total cell lysates and nuclear fractions to understand compartmentalization of signaling .

• Genetic manipulation:

  • Overexpression: Utilize L3MBTL4 overexpression models, similar to the transgenic rat models described in the literature, to observe downstream effects on MAPK pathway components .

  • Knockdown/Knockout: siRNA or CRISPR-Cas9 approaches to reduce L3MBTL4 expression and monitor effects on MAPK pathway activation.

• Pharmacological inhibition: Apply specific inhibitors of p38MAPK (e.g., SB203580), JNK (e.g., SP600125), or MEK/ERK (e.g., U0126) in L3MBTL4-overexpressing systems to identify which MAPK branch is most critical for L3MBTL4-mediated effects .

• LTBP1 interaction studies: Since L3MBTL4 has been shown to down-regulate LTBP1, and this appears to be mechanistically linked to MAPK activation, experiments combining L3MBTL4 manipulation with LTBP1 rescue or depletion can help elucidate the pathway connections .

• Transcriptional profiling: RNA-seq analysis comparing control and L3MBTL4-overexpressing cells, with and without MAPK pathway inhibitors, to identify gene expression changes dependent on both L3MBTL4 and MAPK signaling.

• Biological readouts: Measure vascular smooth muscle cell proliferation, migration, and hypertrophy as functional outcomes of L3MBTL4-mediated MAPK activation .

How can I validate the specificity of an L3MBTL4 antibody for my research?

Validating the specificity of an L3MBTL4 antibody is crucial for ensuring reliable research results. A comprehensive validation approach should include:

• Western blot validation:

  • Compare detection in positive control tissues (e.g., mouse testis) versus negative control tissues

  • Verify the observed molecular weight matches the expected 75-80 kDa range

  • Perform peptide competition assays where the antibody is pre-incubated with excess immunizing peptide

• Genetic manipulation controls:

  • Test antibody in samples with L3MBTL4 overexpression and compare to wild-type controls

  • Test in L3MBTL4 knockdown/knockout samples to confirm signal reduction

• Cross-reactivity assessment:

  • Test the antibody against recombinant proteins of closely related family members (other L3MBTL proteins)

  • Evaluate species cross-reactivity if working with multiple model systems

• Multiple application validation:

  • If using the antibody for IHC, confirm that the staining pattern matches WB results from the same tissues

  • For nuclear proteins like L3MBTL4, confirm appropriate nuclear localization in IHC/ICC applications

• Independent antibody comparison:

  • When possible, compare results from at least two independent antibodies targeting different epitopes of L3MBTL4

• Mass spectrometry validation:

  • For definitive validation, perform immunoprecipitation followed by mass spectrometry identification of the pulled-down proteins

What are the known downstream targets of L3MBTL4 and how can they be studied?

Research has identified several downstream targets of L3MBTL4, with LTBP1 (latent transforming growth factor-β binding protein 1) being a key direct target. To study these targets and identify new ones, researchers can employ these approaches:

• Chromatin immunoprecipitation (ChIP) analysis:

  • ChIP-seq using L3MBTL4 antibodies has successfully identified 3,289 peaks in human aortic smooth muscle cells, with 1,362 successfully mapped to genes

  • These genes were distributed across exonic, intronic, upstream, intergenic, and downstream regions

• Gene Ontology analysis of ChIP-seq data:

  • Identified targets can be analyzed for enrichment in biological processes, cellular components, and molecular functions

  • This approach has helped identify L3MBTL4's role in regulating genes involved in vascular function

• Validation of direct targets:

  • qPCR to confirm transcriptional changes of putative targets

  • ChIP-qPCR to validate specific binding sites

  • For LTBP1 specifically, ChIP showed higher abundance in immunoprecipitated samples compared to control IgG

• Functional analysis of target regulation:

  • Reporter gene assays with the promoter regions of target genes

  • Site-directed mutagenesis of putative L3MBTL4 binding sites

  • Expression analysis in tissues from L3MBTL4 transgenic animals (e.g., repressed transcription activities of LTBP1 were confirmed in blood vessels of L3MBTL4 transgenic rats)

• Pathway analysis:

  • Since L3MBTL4 targets LTBP1 and affects MAPK signaling, studying the phosphorylation status of p38MAPK and JNK provides insight into downstream functional consequences

  • siRNA knockdown of LTBP1 can be used to confirm its role in mediating L3MBTL4's effects on MAPK activation

How does L3MBTL4 expression differ between normal and hypertensive models?

Significant differences in L3MBTL4 expression between normal and hypertensive models have been documented:

• Animal model comparisons:

  • Spontaneously hypertensive rats (SHRs) show more abundant L3MBTL4 mRNA and protein expression in blood vessels compared to normotensive Wistar-Kyoto rats (WKYs)

  • This differential expression pattern was validated in multiple rat cohorts using both qPCR and Western blotting techniques

• Tissue-specific expression differences:

  • The upregulation of L3MBTL4 in hypertensive models is particularly pronounced in vascular tissues

  • Within blood vessels, L3MBTL4 is predominantly expressed in the medial layer, co-localizing with α-actin (a marker for smooth muscle cells)

• Recommended experimental approaches for studying expression differences:

  • Quantitative PCR using validated primers for L3MBTL4

  • Western blotting with standardized loading controls

  • Immunohistochemistry to visualize tissue distribution differences

  • Double immunofluorescence staining with cell-type markers to identify specific cellular expression patterns

• Functional consequences of differential expression:

  • Transgenic rats with ubiquitous overexpression of L3MBTL4 exhibit significantly elevated blood pressure compared to wild-type controls

  • These animals also show increased thickness of the vascular media layer and cardiac hypertrophy, mirroring the pathology observed in spontaneous hypertension

What are the best co-immunoprecipitation conditions for studying L3MBTL4 interactions?

For effective co-immunoprecipitation (Co-IP) studies of L3MBTL4 interactions, the following optimized conditions are recommended:

• Cell/tissue preparation:

  • For vascular tissue: Finely mince fresh tissue and homogenize in ice-cold non-denaturing lysis buffer

  • For cultured cells: Human aortic smooth muscle cells (HASMCs) have been successfully used for L3MBTL4 interaction studies

• Lysis buffer composition:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation-dependent interactions)

• Nuclear extraction (recommended for nuclear proteins like L3MBTL4):

  • First isolate nuclei using hypotonic buffer followed by nuclear extraction buffer

  • This improves signal-to-noise ratio for nuclear protein interactions

• Pre-clearing:

  • Pre-clear lysates with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding

• Antibody incubation:

  • Use 2-5 μg of L3MBTL4 antibody per 500 μg of protein lysate

  • Incubate overnight at 4°C with gentle rotation

• Bead capture:

  • Add pre-washed Protein A/G beads

  • Incubate for 2-4 hours at 4°C with gentle rotation

• Washing conditions:

  • Perform 4-6 washes with washing buffer containing reduced detergent concentration

  • Final wash should be with buffer without detergent

• Controls:

  • IgG control is essential (has been used in previous L3MBTL4 studies)

  • Input samples (5-10% of pre-IP lysate)

  • Reverse Co-IP when antibodies for interaction partners are available

• Elution and detection:

  • Elute with SDS sample buffer at 95°C for 5 minutes

  • Analyze by Western blotting for both L3MBTL4 and suspected interaction partners

  • Consider mass spectrometry for unbiased identification of interaction partners

How might L3MBTL4 study contribute to understanding hypertension mechanisms?

L3MBTL4 research provides several critical insights into hypertension mechanisms that expand our understanding of this complex disease:

• Genetic susceptibility:

  • Genome-wide association studies have identified L3MBTL4 as a novel susceptibility gene for hypertension (rs403814, P = 6.128 × 10^-9)

  • This finding helps explain some of the previously unaccounted genetic contribution to blood pressure variation

• Vascular remodeling pathways:

  • L3MBTL4 overexpression leads to vascular remodeling characterized by increased medial-to-lumen area ratio, a hallmark of hypertensive vascular changes

  • This provides a mechanistic link between genetic factors and structural changes in blood vessels

• Molecular signaling mechanisms:

  • L3MBTL4 regulates the MAPK signaling pathway through suppression of LTBP1

  • Activation of p38MAPK and JNK appears critical for the vascular effects of L3MBTL4

  • This elucidates a previously unrecognized signaling axis contributing to hypertension

• Experimental approaches for hypertension research:

  • L3MBTL4 transgenic models provide new tools for studying hypertension development

  • The identification of L3MBTL4 targets through ChIP-seq offers new candidate genes for hypertension research

• Therapeutic implications:

  • L3MBTL4 represents a potential novel target for antihypertensive therapies

  • The LTBP1-MAPK axis modulated by L3MBTL4 could be targeted pharmacologically

  • Understanding this pathway may help explain the variable efficacy of existing MAPK-targeting drugs in hypertension

What experimental controls should be included when studying L3MBTL4 in vascular remodeling?

When investigating L3MBTL4's role in vascular remodeling, comprehensive controls are essential for reliable interpretation:

• Animal model controls:

  • Age-matched wild-type controls for transgenic L3MBTL4 overexpression models

  • Littermate controls to minimize genetic background variation

  • Sham-operated controls for surgical models of hypertension

  • Both spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs) as comparative models

• Molecular controls:

  • Empty vector controls for L3MBTL4 overexpression studies

  • Non-targeting siRNA/shRNA for knockdown experiments

  • IgG controls for immunoprecipitation and ChIP experiments

  • Multiple housekeeping genes/proteins as loading controls for expression studies

• Pathway validation controls:

  • MAPK pathway inhibitors (p38MAPK inhibitor SB203580, JNK inhibitor SP600125) to confirm signaling specificity

  • Rescue experiments with LTBP1 expression in L3MBTL4-overexpressing systems

  • LTBP1 siRNA to confirm its role in mediating L3MBTL4 effects

• Histological controls:

  • Multiple vascular beds to determine tissue specificity of effects

  • Time-course analyses to distinguish primary from secondary effects

  • Morphometric measurements with blinded analysis to prevent bias

  • Multiple staining methods to confirm vascular remodeling (H&E, Masson's trichrome, elastin staining)

• Physiological controls:

  • Multiple blood pressure measurement techniques (tail-cuff, telemetry)

  • Assessment at different times of day to account for circadian variations

  • Measurements under both resting and stressed conditions

What techniques can be used to study the interaction between L3MBTL4 and LTBP1?

The interaction between L3MBTL4 and LTBP1 is a key mechanistic component of L3MBTL4's role in vascular biology. Several complementary techniques can be employed to study this interaction:

• Chromatin immunoprecipitation (ChIP):

  • ChIP using L3MBTL4 antibodies has successfully shown association with the LTBP1 gene

  • ChIP-seq identified LTBP1 among the 1,362 genes associated with L3MBTL4 binding

  • ChIP-qPCR can be used to quantify the specific binding to LTBP1 regulatory regions

  • These experiments showed higher abundance of LTBP1 in L3MBTL4-immunoprecipitated samples compared to control IgG

• Gene expression analysis:

  • qRT-PCR to quantify LTBP1 mRNA levels in response to L3MBTL4 manipulation

  • Western blotting to assess LTBP1 protein levels

  • In vivo validation in transgenic models (repressed transcription activities of LTBP1 were confirmed in blood vessels of L3MBTL4 transgenic rats)

• Reporter gene assays:

  • Luciferase reporter constructs containing LTBP1 promoter regions

  • Site-directed mutagenesis of putative L3MBTL4 binding sites to identify critical regulatory elements

• Functional validation:

  • siRNA knockdown of LTBP1 to mimic L3MBTL4 effects (has been shown to increase p38MAPK and JNK phosphorylation)

  • LTBP1 overexpression in L3MBTL4-overexpressing systems to determine if it rescues the phenotype

• Protein binding studies:

  • If direct protein-protein interaction is suspected, co-immunoprecipitation with antibodies against both proteins

  • Proximity ligation assays to detect close association in cellular contexts

  • In vitro binding assays with recombinant proteins if direct binding is hypothesized

How can chromatin immunoprecipitation be optimized for L3MBTL4 studies?

Optimizing chromatin immunoprecipitation (ChIP) for L3MBTL4 studies requires careful attention to several critical parameters:

• Starting material:

  • Human aortic smooth muscle cells (HASMCs) have been successfully used for L3MBTL4 ChIP-seq studies

  • Fresh tissue from relevant vascular beds (aorta, resistance vessels) can be used for in vivo studies

  • 10⁶-10⁷ cells per ChIP reaction is recommended for optimal results

• Crosslinking conditions:

  • For nuclear proteins like L3MBTL4, standard formaldehyde crosslinking (1% for 10 minutes at room temperature) is typically sufficient

  • Dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde may improve results for challenging interactions

• Chromatin fragmentation:

  • Sonication should aim for fragments of 200-500 bp for optimal resolution

  • Enzymatic digestion with micrococcal nuclease is an alternative for sensitive epitopes

  • Verify fragmentation efficiency by agarose gel electrophoresis before proceeding

• Antibody selection and validation:

  • Use ChIP-grade antibodies specifically validated for this application

  • Pre-clear chromatin with protein A/G beads to reduce background

  • Include an IgG control to assess non-specific binding

• Washing conditions:

  • Implement increasingly stringent washing steps to reduce background

  • Include a high-salt wash to disrupt non-specific ionic interactions

• Elution and reversal of crosslinks:

  • Elute at 65°C with appropriate elution buffer

  • Reverse crosslinks overnight at 65°C with proteinase K digestion

• Data analysis for ChIP-seq:

  • Use appropriate peak-calling algorithms (e.g., MACS2)

  • Annotate peaks to genomic features using databases like UCSC

  • Perform Gene Ontology analyses on identified targets to understand biological relevance

• Validation of targets:

  • Confirm key targets like LTBP1 using ChIP-qPCR

  • Compare binding patterns across different cellular conditions (e.g., normal vs. stress conditions)