LRRC42 Antibody

What are the optimal applications for LRRC42 antibodies in cancer research?

LRRC42 antibodies are valuable tools in several key cancer research applications. For expression studies, these antibodies are effective in Western blot analysis, as demonstrated in hepatocellular carcinoma cell lines (HCCLM3, Huh7, and HepG2) compared to normal hepatocytes (LO2) . Immunohistochemistry applications are supported by data from the Human Protein Atlas (HPA) database, which reveals LRRC42's predominant expression in the nucleoplasm of A-431, PC-3, and U2OS cell lines . For functional studies, LRRC42 antibodies can be employed in immunoprecipitation assays to study its interactions with other proteins, such as ubiquitin-specific enzyme 7 (USP7), which stabilizes LRRC42 through deubiquitination . In cellular localization studies, immunofluorescence techniques using LRRC42 antibodies help confirm its subcellular distribution and potential colocalization with pathway components.

How does LRRC42 expression vary across different cancer types?

LRRC42 expression exhibits notable variation across cancer types, making antibody-based detection methods crucial for characterization. Comprehensive RNA-seq analysis from The Cancer Genome Atlas (TCGA) database has revealed significant upregulation of LRRC42 in multiple tumor tissues compared to their normal counterparts . Specifically, bladder cancer, breast cancer, cholangiocarcinoma, colorectal cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, liver cancer, lung adenocarcinoma, and skin melanoma all show elevated LRRC42 expression . Conversely, kidney chromophobe and thyroid carcinoma display significant downregulation of LRRC42, suggesting cancer-specific regulatory mechanisms . At the single-cell level, LRRC42 shows high expression in specific cell subpopulations such as Tproif cells of breast, colorectal, esophageal, liver cancers, and nasopharyngeal carcinoma, as well as in monocytes/macrophages of glioma and nasopharyngeal carcinoma . This complex expression pattern necessitates careful selection of positive and negative control tissues when validating LRRC42 antibodies.

How should I design experiments to validate LRRC42 antibody specificity?

When validating LRRC42 antibody specificity, a multi-faceted approach is essential. Begin with Western blot analysis using cell lines known to express different levels of LRRC42, such as hepatocellular carcinoma cell lines (HCCLM3, Huh7, HepG2) compared to normal hepatocytes (LO2) . A specific antibody should detect a single band of the expected molecular weight, with band intensity correlating with known expression levels. Include a knockdown validation by comparing Western blot results between wild-type cells and those treated with LRRC42-targeting shRNAs (as demonstrated in the research with shLRRC42-1, which showed substantial knockdown efficiency) . Immunoprecipitation followed by mass spectrometry can provide additional confirmation of antibody specificity. For immunohistochemistry applications, validate using tissue microarrays containing multiple cancer types with known LRRC42 expression profiles, particularly comparing LRRC42-high cancers (BLCA, BRCA, LIHC) with LRRC42-low cancers (KICH, THCA) . Finally, recombinant expression systems overexpressing tagged LRRC42 can serve as positive controls, while CRISPR/Cas9 knockout cell lines provide definitive negative controls.

What controls should be included when using LRRC42 antibodies for immunohistochemistry?

When conducting immunohistochemistry with LRRC42 antibodies, comprehensive controls are critical for result interpretation. Based on LRRC42's expression patterns, include positive tissue controls from cancers known to overexpress LRRC42, such as liver hepatocellular carcinoma, breast cancer, or bladder cancer . Negative tissue controls should include kidney chromophobe or thyroid carcinoma tissues, which exhibit downregulated LRRC42 expression . Technical negative controls (primary antibody omission) are essential to assess non-specific binding of secondary antibodies. Peptide competition assays, where the antibody is pre-incubated with excess LRRC42 peptide before tissue application, help confirm binding specificity. Since LRRC42 localizes predominantly to the nucleoplasm according to Human Protein Atlas data, nuclear staining should be prominent in positive samples . For validation studies, parallel staining with two different LRRC42 antibodies recognizing distinct epitopes provides additional confidence. When possible, include tissues from experimental models with LRRC42 knockdown (using validated shRNAs like shLRRC42-1) as biological negative controls . Finally, consider dual immunofluorescence labeling to confirm colocalization with known nuclear markers to validate LRRC42's reported nucleoplasmic localization.

How can I optimize immunofluorescence protocols for LRRC42 subcellular localization studies?

Optimizing immunofluorescence protocols for LRRC42 subcellular localization requires careful attention to fixation, permeabilization, and antibody conditions. Since LRRC42 is predominantly expressed in the nucleoplasm according to Human Protein Atlas data, optimal nuclear preservation is essential . Begin with 4% paraformaldehyde fixation for 15-20 minutes to maintain structural integrity while preserving epitope accessibility. For permeabilization, use 0.2-0.5% Triton X-100 for nuclear proteins like LRRC42, adjusting concentration based on cell type. Antigen retrieval may be necessary; test both heat-induced (citrate buffer, pH 6.0) and enzymatic methods to determine optimal epitope exposure. Blocking should include both serum (5-10%) and BSA (1-3%) to minimize non-specific binding. For antibody incubation, extend primary antibody (LRRC42) treatment to overnight at 4°C at optimized dilutions (typically 1:100-1:500), followed by fluorophore-conjugated secondary antibody incubation for 1-2 hours at room temperature. Include DAPI or Hoechst nuclear counterstain to confirm nuclear localization. For co-localization studies, consider dual staining with markers of nuclear subcompartments to precisely map LRRC42's distribution within the nucleoplasm. Confocal microscopy with Z-stack acquisition will provide detailed three-dimensional information about LRRC42's nuclear distribution. Validate observations with super-resolution microscopy techniques when available for nanoscale localization precision.

How can LRRC42 antibodies be used to study the tumor immune microenvironment?

LRRC42 antibodies provide valuable tools for investigating interactions between this protein and the tumor immune microenvironment. Multiplex immunofluorescence combining LRRC42 antibodies with immune cell markers can map spatial relationships between LRRC42-expressing tumor cells and infiltrating immune populations. Research has revealed significant correlations between LRRC42 expression and immune cell infiltration, particularly dendritic cells, macrophages, and neutrophils across multiple tumor types . In hepatocellular carcinoma specifically, LRRC42 shows robust co-expression correlation with Th2 cells (R = 0.375, p < 0.001) . Flow cytometry using LRRC42 antibodies can quantify expression levels in disaggregated tumor samples while simultaneously characterizing immune cell populations. For mechanistic studies, chromatin immunoprecipitation (ChIP) using LRRC42 antibodies can identify potential transcriptional regulation of immune-related genes. Co-immunoprecipitation experiments may reveal direct protein interactions between LRRC42 and immune checkpoint molecules, as significant associations between LRRC42 expression and multiple immune checkpoints have been observed in bladder cancer, low-grade glioma, and liver hepatocellular carcinoma . Moreover, examining LRRC42 expression in sorted immune cell populations from the tumor microenvironment could provide insights into its potential role in immune cell function within tumors.

What methodologies are most effective for studying LRRC42's role in specific signaling pathways?

Investigating LRRC42's role in signaling pathways requires sophisticated methodological approaches centered around high-quality antibodies. Co-immunoprecipitation using LRRC42 antibodies followed by mass spectrometry has revealed its interaction with ubiquitin-specific enzyme 7 (USP7), which stabilizes LRRC42 through deubiquitination and enhances Wnt/β-catenin signaling activity . For studying pathway activation states, proximity ligation assays (PLA) using LRRC42 antibodies paired with antibodies against pathway components can visualize and quantify protein interactions at subcellular resolution. LRRC42 has shown significant correlations with key cancer hallmarks and signaling pathways including MYC targets, mTORC1 signaling, interferon alpha response, E2F targets, and epithelial-mesenchymal transition (EMT) . ChIP-seq using LRRC42 antibodies can map its genome-wide binding profile and identify potential transcriptional targets within these pathways. For functional pathway analysis, combine LRRC42 antibody-based protein detection with reporter assays for specific pathways like Wnt/β-catenin signaling. Phospho-specific antibodies against downstream pathway components should be used alongside LRRC42 antibodies in Western blots following LRRC42 knockdown or overexpression to track signaling cascade dynamics. Co-expression analysis from the TCGA-LIHC cohort has identified genes significantly correlated with LRRC42 that are enriched in pathways such as the spliceosome, cell cycle, and nucleocytoplasmic transport .

How can single-cell techniques be combined with LRRC42 antibodies to advance cancer research?

Combining single-cell techniques with LRRC42 antibodies offers unprecedented resolution for understanding its heterogeneous expression and function in the tumor microenvironment. Single-cell RNA sequencing data from the TISCH2 database has already revealed high LRRC42 expression in specific cell subpopulations across different cancers, including Tproif cells and malignant cells in various tumor types . For protein-level analysis, mass cytometry (CyTOF) incorporating LRRC42 antibodies enables simultaneous measurement of dozens of proteins at single-cell resolution, allowing correlation of LRRC42 expression with cell type, activation state, and other pathway markers. Imaging mass cytometry further adds spatial context to these relationships within the tumor architecture. For in situ detection, multiplexed immunofluorescence using LRRC42 antibodies coupled with immune cell markers and signaling pathway components can visualize expression heterogeneity within intact tissues. Single-cell Western blot techniques may be adapted with LRRC42 antibodies to quantify protein levels in rare cell populations. For functional analysis, FACS sorting of cells based on LRRC42 expression followed by single-cell RNA-seq or ATAC-seq can reveal transcriptional and epigenetic differences between LRRC42-high and LRRC42-low populations. This approach is particularly valuable given the observed correlation between LRRC42 and genes involved in mRNA processing and cell cycle regulation .

What are the optimal knockdown approaches for functional validation of LRRC42?

Effective knockdown approaches for functional validation of LRRC42 require careful design and comprehensive validation using LRRC42 antibodies. RNA interference has proven effective, with shRNA-mediated knockdown successfully demonstrating LRRC42's role in hepatocellular carcinoma . When designing shRNAs targeting LRRC42, create multiple constructs targeting different regions of the transcript, as demonstrated in the referenced study which generated three shRNAs with shLRRC42-1 showing the highest knockdown efficiency . CRISPR/Cas9 approaches offer an alternative for complete gene knockout, designing gRNAs targeting early exons to ensure functional disruption. For inducible systems, consider doxycycline-inducible shRNA or CRISPRi to enable temporal control over LRRC42 depletion. Regardless of the knockdown method, Western blot validation using LRRC42 antibodies is essential to confirm protein-level reduction, as performed in Huh7 and HCCLM3 cell lines . qRT-PCR should complement protein analysis to verify transcript reduction. For knockdown phenotype characterization, functional assays should include proliferation (CCK-8, BrdU), migration (scratch assay), and invasion (Transwell) assessments, all of which demonstrated significant inhibition following LRRC42 knockdown in hepatocellular carcinoma cells . Rescue experiments reintroducing shRNA-resistant LRRC42 variants can confirm knockdown specificity. For pathway analysis, combine knockdown with Western blot detection of phosphorylated pathway components to track signaling alterations.

How should I design experiments to investigate LRRC42's role in tumor cell migration and invasion?

Designing experiments to investigate LRRC42's role in tumor cell migration and invasion requires multiple complementary approaches and careful validation with LRRC42 antibodies. Based on findings that LRRC42 knockdown significantly inhibits migration and invasion in hepatocellular carcinoma cell lines, begin with in vitro scratch wound healing assays to measure collective cell migration following LRRC42 modulation . Transwell migration assays without Matrigel coating provide quantitative data on single-cell chemotactic migration, while Transwell invasion assays with Matrigel coating assess invasive capacity through extracellular matrix . Real-time cell analysis systems can provide continuous monitoring of migration/invasion dynamics. For mechanistic insights, perform Western blot analysis using antibodies against LRRC42 and EMT markers (E-cadherin, N-cadherin, vimentin) following LRRC42 knockdown or overexpression, as LRRC42 exhibits robust correlations with various EMT molecules including MTHFD2, SLC3A2, STAT1, and HIF1A . Immunofluorescence microscopy using LRRC42 antibodies can visualize its localization during migration/invasion and potential colocalization with cytoskeletal components. For advanced studies, 3D spheroid invasion assays more closely mimic in vivo conditions, while microfluidic devices can create controlled chemotactic gradients. Live-cell imaging with fluorescently-tagged LRRC42 constructs complements antibody-based endpoint analyses by tracking dynamic localization during migration. In vivo metastasis assays using LRRC42-knockdown cells provide the ultimate validation of its role in invasion and metastasis.

What methodological considerations are important for studying LRRC42's relationship with autophagy and pyroptosis?

Investigating LRRC42's relationship with autophagy and pyroptosis requires specialized methodological approaches centered around high-quality antibodies. Correlation analyses have revealed significant associations between LRRC42 expression and various autophagy-related factors across multiple tumor types . For autophagy studies, Western blot analysis using LRRC42 antibodies alongside markers LC3-I/II, p62/SQSTM1, and Beclin-1 following LRRC42 modulation can reveal functional relationships. Autophagic flux assays using chloroquine or bafilomycin A1 with LRRC42 knockdown/overexpression help distinguish between increased autophagosome formation versus decreased clearance. Immunofluorescence microscopy with LRRC42 and LC3 antibodies can visualize potential colocalization with autophagosomes. For pyroptosis investigations, LRRC42 has shown notable correlations with diverse pyroptosis-related molecules in tumors including bladder cancer, colorectal cancer, low-grade glioma, and liver hepatocellular carcinoma . Western blot detection of pyroptosis markers (GSDMD, cleaved caspase-1, IL-1β, IL-18) following LRRC42 modulation can reveal regulatory relationships. LDH release assays and propidium iodide uptake following LRRC42 knockdown quantify pyroptotic cell death. Co-immunoprecipitation using LRRC42 antibodies can identify direct interactions with pyroptosis pathway components. For live-cell imaging, combine LRRC42-GFP constructs with fluorescent pyroptosis indicators like GSDMD-mCherry to track dynamics during cell death. These methodologies should be applied across multiple cell lines to account for context-dependent effects.

What are common challenges and solutions when using LRRC42 antibodies for Western blot analysis?

Western blot analysis with LRRC42 antibodies presents several technical challenges that require methodical troubleshooting. Background signal issues can be addressed by optimizing blocking conditions (try 5% non-fat milk versus 3-5% BSA) and increasing washing stringency with higher salt TBST (0.1-0.3% Tween-20). When no signal is detected, verify LRRC42 expression in your cell line, as expression varies significantly between cancer types and cell lines . Hepatocellular carcinoma cell lines (HCCLM3, Huh7, HepG2) show high expression while normal hepatocytes (LO2) have lower levels, making them good positive and comparative controls . Multiple bands may indicate degradation products, alternatively spliced variants, or post-translational modifications; address by using fresh samples with complete protease inhibitors and comparing multiple LRRC42 antibodies targeting different epitopes. For quantification challenges, normalize to appropriate loading controls and validate with known LRRC42-high and LRRC42-low cell lines. Since LRRC42 is predominantly localized to the nucleoplasm, nuclear extraction protocols may provide enriched samples for detection . Denaturation conditions can significantly impact detection; test both reducing and non-reducing conditions, as well as different denaturation temperatures (70°C vs. 95°C). For membrane transfer optimization, adjust transfer conditions based on LRRC42's molecular weight (longer transfer times for larger proteins). Finally, antibody dilution and incubation time should be systematically titrated, typically starting at manufacturer's recommendations and adjusting based on signal-to-noise ratio.

How can I optimize LRRC42 immunoprecipitation for studying protein-protein interactions?

Optimizing LRRC42 immunoprecipitation for studying protein-protein interactions requires careful consideration of experimental conditions to preserve physiologically relevant interactions. LRRC42 has been shown to interact with ubiquitin-specific enzyme 7 (USP7), which stabilizes it through deubiquitination . Begin by selecting cell lysis buffers that balance efficient extraction with interaction preservation; test NP-40 (0.5-1%) for milder extraction versus RIPA for more stringent conditions. Include protease and phosphatase inhibitors to prevent degradation and preserve post-translational modifications. For nuclear proteins like LRRC42, which predominantly localizes to the nucleoplasm, nuclear extraction protocols may increase yield . Pre-clearing lysates with protein A/G beads reduces non-specific binding. When selecting LRRC42 antibodies, choose those validated for immunoprecipitation applications and consider using multiple antibodies against different epitopes to confirm interactions. For the immunoprecipitation procedure, optimize antibody amounts (typically 1-5 μg) and incubation conditions (4°C overnight with gentle rotation). Washing stringency affects interaction detection; use a stepwise approach with increasingly stringent washes. For elution, consider native elution with peptide competition for downstream functional assays versus denaturing elution for mass spectrometry. Cross-linking antibodies to beads can prevent heavy/light chain interference in Western blot detection. For detecting transient or weak interactions, consider chemical crosslinking prior to cell lysis or proximity labeling approaches like BioID or APEX2 fused to LRRC42. Reciprocal immunoprecipitations (e.g., with USP7 antibodies) provide stronger evidence for genuine interactions.

What quality control measures should be implemented for LRRC42 antibody validation?

Comprehensive quality control for LRRC42 antibody validation requires a multi-parameter approach to ensure specificity and reproducibility. Begin with basic Western blot validation using positive control samples from cell lines with known LRRC42 expression, such as hepatocellular carcinoma lines (HCCLM3, Huh7, HepG2), which show high LRRC42 levels compared to normal hepatocytes (LO2) . Implement genetic knockout/knockdown controls by comparing detection in wild-type cells versus those treated with validated LRRC42-targeting shRNAs, such as shLRRC42-1, which demonstrated substantial knockdown efficiency in previous research . For immunohistochemistry applications, conduct parallel staining across multiple tissue types reflecting LRRC42's varied expression pattern, including positive controls (bladder cancer, liver cancer) and negative controls (kidney chromophobe) . Peptide competition assays, where the antibody is pre-incubated with excess LRRC42 peptide/protein, should eliminate specific staining. Apply orthogonal detection methods by correlating antibody-based detection with RNA-expression data from qPCR or RNA-seq. Cross-validate with multiple LRRC42 antibodies targeting different epitopes to confirm consistent detection patterns. For lot-to-lot consistency, maintain reference samples and establish acceptance criteria for new antibody batches. Document subcellular localization patterns, which should show predominant nucleoplasmic staining based on Human Protein Atlas data . Finally, apply application-specific validation; for example, if using the antibody for ChIP-seq, validate enrichment at expected genomic loci with appropriate controls.

What are promising approaches for targeting LRRC42 in cancer therapeutics?

Targeting LRRC42 in cancer therapeutics represents a promising avenue for research, with several approaches warranting investigation. Functional validation through LRRC42 knockdown has demonstrated significant inhibition of cell proliferation, migration, and invasion in hepatocellular carcinoma cell lines, supporting its potential as a therapeutic target . Antibody-drug conjugates (ADCs) utilizing LRRC42 antibodies conjugated to cytotoxic payloads could deliver targeted therapy to LRRC42-expressing tumors, particularly those with high expression like bladder cancer, breast cancer, and liver cancer . Proteolysis-targeting chimeras (PROTACs) targeting LRRC42 for degradation offer an alternative approach, potentially disrupting its interaction with USP7, which stabilizes LRRC42 through deubiquitination . For immunotherapy applications, LRRC42's significant correlations with immune checkpoints in bladder cancer, low-grade glioma, and liver hepatocellular carcinoma suggest potential combination strategies . Small molecule inhibitors targeting the interaction between LRRC42 and key pathway components could be developed through structure-based drug design, though detailed structural information may be required. RNA interference-based therapeutics (siRNA, miRNA) delivered via nanoparticles could achieve LRRC42 knockdown in vivo, building on successful in vitro results with shRNA-mediated knockdown . Finally, considering LRRC42's correlation with autophagy and pyroptosis-related molecules, combination approaches targeting these processes alongside LRRC42 inhibition may enhance therapeutic efficacy .

What future research directions would advance our understanding of LRRC42 biology?

Future research to advance our understanding of LRRC42 biology should leverage antibody-based methods alongside complementary approaches. Comprehensive mechanistic studies should investigate how LRRC42 regulates the Wnt/β-catenin signaling pathway, given its interaction with USP7 and enhancement of pathway activity . Structural biology approaches including cryo-EM and X-ray crystallography using purified LRRC42 (detected with validated antibodies) would elucidate its three-dimensional structure and identify potential binding pockets for drug development. Generation of conditional knockout mouse models with tissue-specific LRRC42 deletion, validated by immunohistochemistry with LRRC42 antibodies, would reveal its roles in development and tissue homeostasis. For cancer progression studies, spatial transcriptomics combined with LRRC42 immunohistochemistry could map expression patterns within the tumor microenvironment in relation to invasive fronts and immune infiltrates. CRISPR screens in LRRC42-high versus LRRC42-low backgrounds may identify synthetic lethal interactions for targeted therapy approaches. Detailed investigation into LRRC42's role in modulating autophagy and pyroptosis is warranted, given significant correlations with related molecules across multiple tumors . Exploration of potential post-translational modifications of LRRC42 using modification-specific antibodies could reveal regulatory mechanisms. Finally, translational research should focus on developing LRRC42-targeting therapeutic strategies based on its demonstrated role in promoting cell proliferation, migration, and invasion in hepatocellular carcinoma .