lrwd1 Antibody

Basic Research Questions

• What is LRWD1 and what are its key functions in cellular processes?

LRWD1 is a multifunctional nuclear protein containing both leucine-rich repeat domains and WD40 repeat domains. It serves several critical cellular functions:

LRWD1 supports DNA replication regulation by recruiting and stabilizing the origin recognition complex (ORC) onto chromatin during G1 phase to establish pre-replication complexes . It is required for proper G1/S transition and is considered an integral part of the DNA replication licensing pathway . Beyond replication, LRWD1 plays a significant role in heterochromatin maintenance by binding to repressive histone marks including H3K9me3, H3K27me3, and H4K20me3 through its WD40 domain . This binding is particularly important for the silencing of major satellite repeats in the genome .

Recent knockout studies have revealed that LRWD1 impacts cell proliferation, as mouse embryonic fibroblasts (MEFs) depleted of LRWD1 display reduced proliferation compared to wild-type cells . Furthermore, while LRWD1 depletion doesn't affect embryonic development, it does lead to retarded postnatal growth in homozygous mutant mice , suggesting an important though non-essential role in developmental processes.

Interestingly, in mouse models, LRWD1 demonstrates testis-specific expression in adult tissues and shows strong localization to the centrosomal region in mature spermatozoa, specifically at the connection between the head and neck , indicating potential specialized functions in spermatogenesis.

• What experimental techniques are most effective for detecting LRWD1 expression in different sample types?

Researchers can employ multiple complementary techniques to detect LRWD1 expression, each offering unique advantages:

Western Blotting (WB): This technique provides quantitative assessment of LRWD1 protein levels. Select antibodies targeting specific epitopes based on your research focus - N-terminal antibodies (amino acids 1-30) work well for detecting full-length protein , while domain-specific antibodies may be needed for studies involving truncated variants. When analyzing mouse samples, remember that LRWD1 shows testis-specific expression in adult tissues .

Immunohistochemistry (IHC-P): For tissue sections, polyclonal antibodies targeting amino acids 100-200 of human LRWD1 have demonstrated good results . Pre-extraction with 0.5% Triton X-100 before fixation can improve signal-to-noise ratio by removing soluble proteins . This approach is particularly valuable for studying LRWD1's distribution within tissue architecture.

Immunofluorescence (IF): For subcellular localization studies, use antibodies validated for IF applications. Co-staining with heterochromatin markers (H3K9me3, HP1α) or centrosomal markers (γ-tubulin) can provide context for LRWD1 localization . High-resolution confocal microscopy is recommended for detailed localization studies.

Co-immunoprecipitation (Co-IP): When studying LRWD1's interactions with proteins like ORC components, use lysis buffers containing 50 mM HEPES-KOH (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, and appropriate protease inhibitors . Multiple washing steps with reduced detergent (0.01% Nonidet P-40) help minimize background while preserving specific interactions.

RT-PCR/Northern blotting: For mRNA detection, design primers spanning exon-exon junctions to avoid genomic DNA contamination. These techniques are particularly important for validating knockout models or assessing tissue-specific expression patterns.

• How does LRWD1 interact with heterochromatin and what methods best detect these interactions?

LRWD1 interacts with heterochromatin through specific molecular mechanisms that can be studied through various experimental approaches:

LRWD1 binds to repressive histone marks through its WD40 domain, showing strong preference for trimethylated marks including H3K9me3, H3K27me3, and H4K20me3 . This binding occurs independently of ORC proteins, though LRWD1 and ORC often function together at heterochromatic regions . Importantly, the recruitment of LRWD1 to pericentric heterochromatin specifically requires H3K9me3, but not H4K20me3 .

For studying these interactions, peptide pulldown assays have proven effective. In this approach, synthetic histone peptides carrying specific methylation marks are used as bait to capture LRWD1 from cell lysates . This can be performed with both wild-type LRWD1 and domain mutants to identify critical interaction regions.

Chromatin immunoprecipitation (ChIP) experiments reveal that LRWD1 binds to heterochromatic regions enriched for repressive marks. When performing ChIP with LRWD1 antibodies, include controls for known heterochromatin regions (major satellites) and euchromatic regions as negative controls. Co-ChIP or sequential ChIP (re-ChIP) can determine if LRWD1 and specific histone marks or ORC proteins co-occupy the same genomic regions.

The functional significance of these interactions can be assessed through LRWD1 depletion studies. Knockout of LRWD1 in MEFs increases expression of epigenetically silenced repetitive elements without significantly affecting protein-coding genes , demonstrating LRWD1's role in maintaining heterochromatin silencing.

Fluorescence microscopy shows LRWD1 co-localization with heterochromatin foci marked by DAPI-dense regions, H3K9me3, or HP1α in the nucleus . For optimal visualization, pre-extract cells with 0.5% Triton X-100 before fixation to remove soluble nuclear proteins.

• What is the tissue and subcellular distribution pattern of LRWD1 expression?

LRWD1 displays distinct tissue-specific and developmental expression patterns with precise subcellular localization:

In adult mice, Northern and Western blot analyses demonstrate that LRWD1 expression is predominantly testis-specific . Within the testis, immunostaining reveals high levels of LRWD1 in primary spermatocytes through to mature spermatozoa, with comparatively weaker signals in spermatogonia . This expression pattern suggests specialized functions during spermatogenesis.

During mouse embryonic development, LRWD1 is ubiquitously expressed throughout most of the developing embryo , indicating broader developmental roles before its expression becomes restricted in adult tissues.

At the subcellular level, LRWD1 displays context-dependent localization patterns. In cultured cells, LRWD1 predominantly localizes to the nucleus, particularly at heterochromatic regions marked by H3K9me3 . This localization is consistent with its role in heterochromatin maintenance and DNA replication regulation.

In mature mouse spermatozoa, LRWD1 shows striking enrichment at the connection region between the head and neck where the centrosome is located . Co-localization and interaction with γ-tubulin, as demonstrated by immunostaining and co-immunoprecipitation, further supports LRWD1's association with the centrosome .

During cell cycle progression, LRWD1 remains chromatin-bound throughout most phases, with particular importance during G1 phase when it helps establish pre-replication complexes . After DNA replication, LRWD1 continues to associate with heterochromatic regions, potentially helping maintain their silenced state.

• What are the structural characteristics of LRWD1 protein and their functional implications?

The LRWD1 protein possesses a distinctive domain architecture that directly relates to its diverse cellular functions:

Mouse LRWD1 consists of 648 amino acids and shares 78.3% sequence identity with human LRWD1 . The protein contains two major structural domains: a leucine-rich repeat (LRR) domain and a WD40 repeat domain. These domains mediate different protein-protein and protein-chromatin interactions.

The WD40 repeat domain forms a β-propeller structure that serves as a critical platform for binding methylated histone marks, particularly H3K9me3, H3K27me3, and H4K20me3 . Mutations in this domain abolish binding to these histone marks and disrupt LRWD1's heterochromatin localization, demonstrating its functional importance.

The N-terminal region (amino acids 1-30) contains epitopes recognized by several LRWD1 antibodies, making it an important target for detection in experimental applications . The middle portion of the protein (amino acids 100-200) contains functional domains involved in protein interactions and is targeted by other antibodies frequently used in research .

LRWD1 interacts with the origin recognition complex (ORC) and is required for ORC2, ORC3, and ORC4 stability . This interaction is essential for LRWD1's role in DNA replication licensing and pre-replication complex assembly.

Various recombinant constructs and truncated versions of LRWD1 have been developed for functional studies, including FLAG-tagged and GFP-tagged variants . These tools allow researchers to investigate domain-specific functions and visualize LRWD1 localization in living cells.

Advanced Research Questions

• What are the optimal experimental conditions for using LRWD1 antibodies in chromatin immunoprecipitation studies?

Successful chromatin immunoprecipitation (ChIP) with LRWD1 antibodies requires careful optimization of multiple parameters:

Table 1: Optimized ChIP Protocol Parameters for LRWD1

When selecting LRWD1 antibodies for ChIP, those targeting the N-terminal region (amino acids 1-30) often provide good results . Avoid antibodies targeting the WD40 domain if possible, as this domain directly interacts with chromatin and may be inaccessible when LRWD1 is bound to heterochromatin.

For heterochromatin studies, include known LRWD1 binding sites such as major satellite repeats as positive controls . Effective sonication is crucial for heterochromatic regions, which can be resistant to shearing due to their compact structure. Verify chromatin fragmentation by agarose gel electrophoresis before proceeding with immunoprecipitation.

Since LRWD1 interacts with ORC proteins, consider sequential ChIP (re-ChIP) to investigate co-occupancy of LRWD1 with ORC components or specific histone marks like H3K9me3 . This approach provides stronger evidence for simultaneous binding of multiple factors to the same genomic regions.

For ChIP-qPCR analysis of repetitive elements, design primers specific to major satellite sequences, as these regions show elevated expression upon LRWD1 depletion . For genome-wide analysis, ChIP-seq may require specialized bioinformatic approaches to analyze repetitive regions of the genome accurately.

• How can LRWD1 knockdown or knockout models be effectively created and validated for functional studies?

Creating reliable LRWD1 depletion models requires strategic approaches and thorough validation:

For transient knockdown studies, siRNA or shRNA approaches targeting different regions of LRWD1 mRNA can be employed. Design multiple siRNAs to ensure specificity and control for off-target effects. Validation should include both mRNA assessment (qRT-PCR) and protein detection (Western blot), with 70-80% reduction typically considered successful knockdown.

For stable genetic models, researchers have successfully employed a gene-trap approach to generate Lrwd1 knockout mice . This technique involves inserting a reporter/selector cassette into an intron of the Lrwd1 gene, disrupting proper transcript processing. When creating similar models, heterozygous animals should be intercrossed to generate homozygous knockouts, with genotyping performed by PCR analysis of genomic DNA.

CRISPR-Cas9 genome editing offers an efficient alternative for generating cell line models. Target guide RNAs to early exons of LRWD1 for complete disruption of protein expression. After clonal selection, validate edited clones by sequencing the target region and confirming protein loss by Western blotting with antibodies targeting different epitopes of LRWD1.

Functional validation of LRWD1 knockdown/knockout should examine specific phenotypes including: 1) Cell proliferation rates, which are typically reduced in LRWD1-depleted cells ; 2) Expression of repetitive elements, particularly major satellite repeats, which show increased expression upon LRWD1 loss ; 3) Chromatin binding of ORC components, which may be destabilized without LRWD1 ; and 4) Cell cycle progression, particularly at the G1/S transition where LRWD1 functions in pre-replication complex assembly .

Rescue experiments expressing wild-type LRWD1 in knockout cells provide strong evidence for phenotype specificity. Consider also expressing domain mutants (e.g., WD40 domain mutants) to dissect the contribution of specific domains to LRWD1 function.

• What is the role of LRWD1 in heterochromatin maintenance during DNA replication?

LRWD1 serves as a critical link between DNA replication and heterochromatin maintenance through several mechanisms:

During DNA replication, the challenge of preserving epigenetic states across cell divisions requires coordination between the replication machinery and chromatin modifiers. LRWD1 (also known as ORCA) plays a pivotal role in this process by interacting with both the origin recognition complex (ORC) and repressive histone marks .

LRWD1 facilitates prereplication complex formation specifically at late-replicating origins, which typically include heterochromatic regions . By binding to H3K9me3 through its WD40 domain, LRWD1 helps recruit ORC to these regions, ensuring their proper replication timing. This mechanism provides a direct link between heterochromatin marks and replication initiation.

The recruitment of LRWD1 to heterochromatin requires H3K9me3 . Once bound, LRWD1 helps maintain heterochromatin silencing, as evidenced by increased expression of major satellite repeats upon LRWD1 depletion . This suggests LRWD1 helps preserve the repressive chromatin state through the disruption caused by DNA replication.

After DNA replication, LRWD1 continues to associate with heterochromatic sites in post-replicated cells , potentially serving as a platform for the re-establishment of repressive histone marks on newly synthesized DNA. This persistent association helps ensure epigenetic inheritance of heterochromatin states across cell divisions.

The dual function of LRWD1 in both DNA replication and heterochromatin maintenance positions it as a key factor in coordinating these processes, ensuring that both genetic and epigenetic information are faithfully transmitted during cell division. This function is particularly important for repetitive DNA elements, which must remain silenced to maintain genomic stability.

• How does LRWD1 contribute to centrosome function and what methods best detect its centrosomal localization?

LRWD1 displays notable centrosomal localization with potential functional implications for cell division:

In mouse spermatozoa, LRWD1 shows strong localization to the connection region between the head and neck where the centrosome is located . This specific localization pattern suggests LRWD1 may have specialized functions in centrosome biology, particularly during spermatogenesis. Co-localization and direct interaction with γ-tubulin, a core centrosomal marker, further supports LRWD1's centrosomal association .

For detecting LRWD1's centrosomal localization, immunofluorescence microscopy with specific sample preparation techniques yields optimal results. Pre-extraction with 0.5% Triton X-100 before paraformaldehyde fixation effectively removes soluble proteins, enhancing visualization of centrosome-bound LRWD1 . Co-staining with established centrosomal markers such as γ-tubulin, centrin, or pericentrin provides necessary reference points.

Biochemical fractionation represents another valuable approach. Isolating centrosome-enriched fractions through sucrose gradient centrifugation followed by Western blotting with LRWD1 antibodies can confirm centrosomal association. Co-fractionation analysis with known centrosomal proteins provides supporting evidence.

For functional studies of LRWD1 at the centrosome, co-immunoprecipitation experiments can identify interacting centrosomal proteins. Using either LRWD1 antibodies to pull down centrosomal proteins or centrosomal protein antibodies to co-precipitate LRWD1 can establish these interactions .

While the precise function of LRWD1 at the centrosome remains to be fully elucidated, its localization pattern and interaction with γ-tubulin suggest potential roles in centrosome organization, centriole duplication, or spindle formation during cell division. The testis-specific expression pattern in adult mice further suggests specialized functions in male germ cell development.

• What are the phenotypic effects of LRWD1 depletion in different cell types and developmental stages?

LRWD1 depletion produces distinct phenotypic consequences depending on cellular context and developmental timing:

During embryonic development, Lrwd1 knockout mice show surprisingly normal progression, suggesting potential compensatory mechanisms during this critical period . Despite LRWD1 being ubiquitously expressed throughout most of the developing mouse embryo, its absence does not prevent embryonic development completion .

At the cellular level, mouse embryonic fibroblasts (MEFs) derived from Lrwd1 knockout mice exhibit reduced proliferation rates compared to wild-type cells . This proliferation defect aligns with LRWD1's known role in DNA replication, particularly in facilitating pre-replication complex formation at origins.

Molecular consequences of LRWD1 depletion include increased expression of epigenetically silenced repetitive elements, particularly major satellite repeats . This derepression demonstrates LRWD1's function in maintaining heterochromatin silencing. Interestingly, LRWD1 loss has minimal effects on protein-coding gene expression , suggesting its silencing function is largely specific to repetitive elements.

Cell cycle analysis reveals that LRWD1 depletion particularly affects the G1/S transition , consistent with its role in origin licensing. The destabilization of ORC components (ORC2, ORC3, and ORC4) observed in LRWD1-depleted cells likely contributes to this cell cycle defect by compromising pre-replication complex assembly.

• How does LRWD1 collaborate with origin recognition complex (ORC) proteins in DNA replication?

LRWD1 and ORC proteins engage in a sophisticated collaboration essential for regulated DNA replication:

LRWD1 (also known as ORCA) functions as a recruitment and stabilization factor for the origin recognition complex. It recruits and stabilizes ORC onto chromatin during G1 phase, particularly at late-replicating origins which often include heterochromatic regions . This recruitment function is crucial for establishing the foundation of DNA replication initiation.

Beyond simple recruitment, LRWD1 appears necessary for the stability of specific ORC subunits. It may be particularly important for maintaining ORC2, ORC3, and ORC4 protein stability , suggesting LRWD1 acts as a molecular scaffold that helps preserve ORC integrity during the extended period from origin licensing to activation.

LRWD1 creates a critical link between chromatin state and replication timing through its ability to bind repressive histone marks. By recognizing H3K9me3, H3K27me3, and H4K20me3 via its WD40 domain , LRWD1 helps coordinate replication timing with heterochromatin state, ensuring proper replication of these specialized genomic regions.

The requirement of LRWD1 for proper G1/S transition indicates its role extends beyond initial recruitment to the timing regulation of replication initiation. This function is particularly significant for heterochromatic regions that typically replicate late in S phase.

After DNA replication completes, LRWD1 and ORC remain associated with certain chromatin regions, particularly heterochromatin . This persistent association likely helps re-establish proper chromatin states on newly synthesized DNA, maintaining epigenetic information through cell division.

To study this collaboration experimentally, techniques such as reciprocal co-immunoprecipitation, chromatin fractionation, and proximity ligation assays provide valuable insights into LRWD1-ORC interactions in different cellular contexts and cell cycle stages.

• What are the best controls to validate LRWD1 antibody specificity in various applications?

Rigorous validation of LRWD1 antibodies requires systematic controls across different experimental applications:

For Western blotting validation, include positive controls from tissues or cells with known LRWD1 expression, such as testis tissue for mouse studies or proliferating cell lines for human samples. The expected molecular weight for LRWD1 is approximately 70-75 kDa. Critical negative controls include lysates from LRWD1 knockout or knockdown cells. When using polyclonal antibodies, peptide competition assays provide additional specificity confirmation - pre-incubation of the antibody with the immunizing peptide should abolish specific bands .

In immunohistochemistry and immunofluorescence applications, compare staining patterns with known expression profiles, such as nuclear/heterochromatin localization in cultured cells or testis-specific expression in mouse tissues . Pre-absorption controls with immunizing peptides and secondary antibody-only controls help distinguish specific signals from background. The subcellular localization should match expected patterns: nuclear with heterochromatin enrichment in cultured cells, or centrosomal localization in spermatozoa .

For chromatin immunoprecipitation, include input DNA samples (typically 5-10% of starting material) to normalize enrichment values. IgG controls from the same species as the LRWD1 antibody identify non-specific binding. ChIP from LRWD1-depleted cells should show significantly reduced signal at target regions, while known LRWD1-binding regions like heterochromatin should show enrichment compared to non-target regions.

When performing immunoprecipitation or co-immunoprecipitation, include input samples representing 5-10% of starting material, IgG controls to identify non-specific interactions, and validation of known LRWD1 interacting proteins such as ORC components . Reciprocal IP with antibodies against interacting partners provides stronger evidence for specific interactions.

Employing multiple antibodies targeting different epitopes of LRWD1 in parallel experiments provides particularly compelling validation. Agreement between antibodies recognizing distinct regions (N-terminal versus middle region) strongly supports result specificity.

• How can researchers distinguish between the different functions of LRWD1 in heterochromatin silencing versus DNA replication?

Differentiating between LRWD1's dual roles requires specialized experimental strategies:

Domain-specific mutants offer powerful tools for separating LRWD1's functions. Generate LRWD1 variants with mutations in the WD40 domain that disrupt histone mark binding while preserving ORC interaction . Similarly, create mutants that maintain histone mark binding but cannot interact with ORC. Express these mutants in LRWD1-depleted cells and assess their ability to rescue different phenotypes - heterochromatin silencing versus replication defects.

Cell cycle synchronization provides temporal separation of LRWD1's functions. Analyze LRWD1 in G1 phase to focus on its pre-replication complex formation role, or examine post-replication periods to isolate its heterochromatin maintenance function. Synchronization methods like thymidine block, nocodazole treatment, or FACS sorting enable precise temporal analysis of LRWD1 activities.

Utilize distinct functional readouts for each role. For heterochromatin silencing, measure expression of repetitive elements (particularly major satellite repeats) by RT-qPCR and assess heterochromatin mark distribution by ChIP. For DNA replication functions, employ BrdU incorporation assays, analyze pre-RC formation by chromatin fractionation of ORC components, or measure replication timing profiles.

ChIP-seq analysis comparing LRWD1 binding sites with known replication origins versus heterochromatin regions can spatially distinguish its different functions. Analyze how LRWD1 binding patterns change across the cell cycle, particularly during G1 (origin licensing) versus post-replication periods (heterochromatin maintenance).

Identify specific protein interaction partners involved in each function through techniques like BioID, IP-MS, or proximity ligation assay. Heterochromatin partners would include HP1 and SUV39H1/2, while replication partners would include ORC, CDC6, CDT1, and MCM proteins. Differential interaction profiles help separate LRWD1's distinct functional roles.

Rapid protein depletion systems like auxin-inducible degradation enable temporal analysis of immediate effects following LRWD1 loss, helping separate direct functions from secondary consequences that develop over longer timeframes.

• What methodological approaches can determine LRWD1 post-translational modifications and their functional significance?

While specific information on LRWD1 post-translational modifications wasn't provided in the search results, researchers can employ these methodological approaches to characterize them:

Mass spectrometry represents the gold standard for identifying PTMs on LRWD1. Immunoprecipitate endogenous or tagged LRWD1 from cells at different cell cycle stages, digest with proteases, and analyze by LC-MS/MS. For phosphorylation studies, employ phospho-enrichment techniques like titanium dioxide (TiO2) chromatography or immobilized metal affinity chromatography (IMAC). Compare PTM profiles across different cellular conditions, particularly during cell cycle progression, to identify regulatory modifications.

Site-specific mutant analysis provides functional insights after identifying modification sites. Generate non-modifiable mutants (e.g., serine-to-alanine for phosphorylation sites) and express in LRWD1-depleted cells to assess functional consequences. Create phosphomimetic mutants (e.g., serine-to-glutamate) to simulate constitutive phosphorylation. Compare the ability of these mutants to rescue specific LRWD1 functions in knockout backgrounds.

Cell cycle synchronization studies reveal temporal patterns of LRWD1 modifications. Synchronize cells at different cell cycle stages using methods like thymidine block (G1/S), nocodazole treatment (M phase), or serum starvation/release (G0/G1). Analyze LRWD1 modification states across the cell cycle using modification-specific antibodies if available, or through mass spectrometry.

Identify the enzymes responsible for LRWD1 modifications through candidate approaches. For phosphorylation, test cell cycle kinases like CDKs, PLKs, or ATM/ATR using specific inhibitors or depletion studies. For other modifications, examine enzymes known to modify nuclear proteins and conduct in vitro modification assays with recombinant LRWD1.

To assess functional impacts, analyze how modifications affect LRWD1 properties including protein stability (cycloheximide chase assays), chromatin binding (chromatin fractionation, ChIP), interaction with partners like ORC (co-IP studies), and subcellular localization (immunofluorescence microscopy).

Generate or obtain antibodies specific to modified forms of LRWD1 once key modification sites are identified. These reagents enable direct visualization and quantification of modified LRWD1 populations in different cellular contexts.

• What methodological strategies enable effective comparison of LRWD1 expression between normal and pathological tissues?

Comparing LRWD1 expression between normal and pathological tissues requires rigorous methodological approaches:

Sample preparation requires careful consideration. Collect matched pairs of normal and pathological tissues when possible to minimize inter-individual variation. For protein analysis, flash-freeze samples immediately after collection to prevent degradation. For histological studies, use standardized fixation protocols (typically 10% neutral buffered formalin) with consistent fixation times to ensure comparable antibody accessibility.

For protein expression analysis, Western blotting offers quantitative comparison when properly controlled. Extract proteins using consistent protocols, verify equal loading with housekeeping proteins like GAPDH or β-actin, and use multiple antibodies targeting different LRWD1 epitopes for confirmation . Densitometric analysis with appropriate normalization enables statistical comparison between sample groups.

Immunohistochemistry provides spatial context for expression differences. Use tissue microarrays when available for high-throughput comparison under identical staining conditions. Implement automated scoring systems to quantify staining intensity and distribution objectively. Co-staining with cell type-specific markers helps identify which cell populations show altered LRWD1 expression within heterogeneous tissues.

RNA expression analysis through RT-qPCR provides a complementary approach. Design primers targeting conserved LRWD1 regions and normalize to multiple reference genes selected for stability in your specific tissue types. RT-qPCR is particularly valuable for validation of findings from broader screening approaches.

Functional correlations strengthen expression comparisons. Assess correlations between LRWD1 levels and proliferation markers (Ki-67, PCNA), heterochromatin markers (H3K9me3, HP1), or clinical parameters. These correlations may provide insights into the biological significance of expression differences.

Through these methodological approaches, researchers can robustly compare LRWD1 expression between normal and pathological states, potentially revealing new insights into disease mechanisms or identifying novel biomarkers.