lrrc3b Antibody

What is LRRC3B and why is it important in research?

LRRC3B (leucine rich repeat containing 3B) is a membrane-localized protein of 259 amino acid residues with a molecular weight of approximately 29.3 kDa. It belongs to the LRRC3 protein family and contains characteristic leucine-rich repeat motifs spanning 20-29 amino acid residues with repetitive hydrophobic residues, particularly leucine . LRRC3B functions as a tumor suppressor gene involved in the anti-tumor immune microenvironment. Its importance in research stems from its roles in immunity, hormone-receptor interactions, cell adhesion, signal transduction, gene expression regulation, and apoptosis. LRRC3B exhibits lower expression in various cancer tissues compared to adjacent normal tissues, making it a promising biomarker for cancer diagnosis, prognosis, and prediction of therapeutic response .

Which experimental models express LRRC3B and where is it primarily localized?

LRRC3B is predominantly expressed in testis, skeletal muscle, and cerebral cortex in humans . The gene has orthologs in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken, enabling comparative studies across different experimental models . Subcellularly, LRRC3B localizes to the membrane with extracellular domain orientation, as revealed by protein topology analyses . It's worth noting that expression levels in cancer cell lines such as H1299 (lung cancer) and Hs578T (breast cancer) are notably low and often difficult to detect using conventional methods like qPCR, which may necessitate more sensitive detection approaches when working with these models .

How can I verify LRRC3B antibody specificity for my experiments?

Verification of LRRC3B antibody specificity requires a multi-faceted approach. First, conduct Western blotting using positive control samples (tissues with known high LRRC3B expression such as testis, skeletal muscle, or cerebral cortex) alongside your samples of interest. The antibody should detect a band at approximately 29.3 kDa corresponding to the canonical LRRC3B protein . Consider including a negative control with LRRC3B knockdown cells.

For immunohistochemistry or immunofluorescence applications, compare staining patterns between normal tissues known to express LRRC3B and cancer tissues where expression is typically reduced. Membranous staining pattern should be observed consistent with the protein's known localization . Additionally, peptide competition assays where the antibody is pre-incubated with the immunizing peptide can further confirm specificity by demonstrating signal abolishment. Due to potential post-translational modifications like glycosylation, slight variations in molecular weight might be observed .

How can LRRC3B antibodies be utilized to study tumor microenvironment interactions?

LRRC3B antibodies serve as crucial tools for investigating tumor microenvironment (TME) dynamics. Multiplex fluorescence immunohistochemistry (mIHC) using LRRC3B antibodies in combination with immune cell markers (CD4, CD8, FOXP3, CD68) enables simultaneous detection of LRRC3B expression and immune cell infiltration within tumor tissues . This approach has revealed that tumors with higher LRRC3B expression exhibit increased infiltration of CD4+ and CD8+ T cells, suggesting LRRC3B's role in modulating anti-tumor immunity .

For more sophisticated analyses, researchers can employ flow cytometry with LRRC3B antibodies to quantify expression levels across different cell populations within the TME. Co-immunoprecipitation studies using LRRC3B antibodies can identify interacting partners involved in immune signaling pathways. When combined with single-cell RNA sequencing data, these approaches provide comprehensive insight into how LRRC3B expression influences the recruitment and activation of various immune cell subtypes, including B cells, CD4+ T cells, CD8+ T cells, and myeloid-derived suppressor cells (MDSCs) .

What methodologies are recommended for studying LRRC3B methylation status in conjunction with protein expression?

A comprehensive approach to studying LRRC3B methylation and protein expression involves integrating multiple methodologies. For methylation analysis, bisulfite sequencing PCR (BSP) targeting the LRRC3B promoter region is recommended, focusing on the 25 CpG sites identified as critical in cancer progression . Alternatively, methylation-specific PCR (MSP) can provide a more accessible approach for screening purposes.

To correlate methylation with protein expression, implement a dual-detection strategy: First, extract DNA and protein from the same sample. After methylation analysis, perform Western blotting with anti-LRRC3B antibody (using protocols as described in the literature with RIPA buffer extraction and appropriate antibody dilutions) . For tissue sections, consider consecutive sections for methylation analysis and immunohistochemistry. This approach has revealed inverse relationships between promoter methylation and protein expression in multiple cancer types including lung, breast, and colorectal cancers .

Which applications are best suited for LRRC3B antibody detection and what are their limitations?

Western Blot and immunofluorescence represent the most widely used and validated applications for LRRC3B antibody detection . Western Blot provides quantitative assessment of LRRC3B protein levels and confirmation of molecular weight, while immunofluorescence enables visualization of subcellular localization and expression patterns within tissue architecture. ELISA also serves as a useful application, particularly for high-throughput screening .

Each method carries limitations researchers should consider. Western Blot may struggle with sensitivity when detecting endogenous LRRC3B in cancer cell lines where expression is notably low, as observed in H1299 and Hs578T cells . Immunohistochemistry applications may face background issues due to LRRC3B's membrane localization potentially obscuring specific signal from non-specific membrane staining. For flow cytometry, the predominantly membrane-localized nature of LRRC3B requires careful optimization of fixation and permeabilization protocols to maintain epitope accessibility without compromising membrane integrity.

To overcome these limitations, consider using signal amplification techniques such as tyramide signal amplification for immunohistochemistry or highly sensitive chemiluminescent substrates for Western Blot. Additionally, validation with multiple antibodies targeting different epitopes of LRRC3B can enhance detection confidence.

What are the recommended protocols for immunoblotting of LRRC3B protein?

For optimal immunoblotting of LRRC3B protein, follow this methodological approach: Lyse cells or tissues in RIPA buffer supplemented with protease inhibitor cocktail. Quantify total protein using BCA Protein Assay Kit (Thermo) . Prepare cell lysates in SDS loading buffer (50 mM TrisHCl pH 6.8, 10% glycerol, 2% SDS, 1% 2-mercaptoethanol, 0.1% bromophenol blue), boil samples, and separate by SDS-PAGE .

Transfer proteins to PVDF membranes using standard wet transfer protocols. Block membranes in Quickblock blocking buffer for 15 minutes at room temperature. Incubate with primary anti-LRRC3B antibody (recommended options include Affinity Biosciences, DF16048) overnight at 4°C . After washing with TBST, incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature. Visualize using high-sensitivity chemiluminescent substrates such as SuperSignal West Femto Maximum Sensitivity Substrate .

For optimal results when working with samples expected to have low LRRC3B expression, load higher protein amounts (50-100 µg), extend primary antibody incubation to 24-48 hours, and use signal enhancers during detection. Include positive control lysates from tissues known to express LRRC3B (testis or skeletal muscle) alongside vinculin as loading control .

How does LRRC3B expression correlate with cancer progression and immune infiltration?

LRRC3B expression exhibits significant negative correlation with cancer progression across multiple tumor types. Higher LRRC3B expression associates with less tumor invasion, less severe tumor staging, and decreased metastatic potential . This pattern has been documented in gastric cancer, renal cancer, colorectal cancer, lung cancer, and breast cancer, where LRRC3B expression is consistently lower in tumor tissue compared to adjacent normal tissue .

Regarding immune infiltration, LRRC3B expression positively correlates with anti-tumor immune cell presence. Tumors with higher LRRC3B expression show increased infiltration of B cells, CD4+ T cells, CD8+ T cells, and antigen-presenting cells, creating a more immunologically favorable microenvironment . This relationship has been validated using multiplexed fluorescence immunohistochemistry, which confirmed higher CD4+/CD8+ T cell infiltration in tumors with elevated LRRC3B expression .

Conversely, LRRC3B downregulation associates with immunosuppressive microenvironments characterized by increased presence of M2 macrophages, myeloid-derived suppressor cells (MDSCs), cancer-associated fibroblasts (CAFs), and regulatory T cells (Tregs) . These findings suggest that LRRC3B functions not only as a direct tumor suppressor but also as a modulator of anti-tumor immunity, potentially explaining its significant impact on patient prognosis.

Can LRRC3B antibodies aid in predicting immunotherapy response, and what methodology should be employed?

LRRC3B antibodies can significantly contribute to predicting immunotherapy response through integration with methylation analysis. The recommended methodological approach involves a two-pronged strategy: First, assess LRRC3B protein expression using immunohistochemistry or Western blotting with specific antibodies . Second, analyze the methylation status of the LRRC3B promoter region, focusing on the 25 CpG sites shown to be clinically relevant .

The methodology for clinical validation should include tissue microarray analysis with anti-LRRC3B antibodies, paired with bisulfite sequencing of the promoter region from the same samples. Correlation with treatment outcomes in immunotherapy cohorts provides the final validation step. This approach has been successfully implemented in public datasets (GSE119144 and GSE72308), confirming that patients with hypomethylated LRRC3B show significantly better response rates to anti-PD-1 treatment .

What challenges might researchers encounter when detecting low LRRC3B expression in cancer cell lines?

Researchers frequently encounter significant challenges when detecting LRRC3B in cancer cell lines due to its characteristically low expression levels. Studies with H1299 (lung cancer) and Hs578T (breast cancer) cell lines revealed LRRC3B levels that were barely detectable by conventional qPCR methods . This low expression creates several technical hurdles that researchers must navigate.

First, standard Western blot protocols may yield weak or undetectable signals. To overcome this limitation, implement protein concentration steps (e.g., immunoprecipitation before Western blotting), extend exposure times, and utilize highly sensitive chemiluminescent substrates designed for low-abundance proteins. Signal amplification techniques such as biotinylated secondary antibodies with streptavidin-HRP can increase detection sensitivity by 10-100 fold.

For immunocytochemistry applications, researcher should employ tyramide signal amplification systems or quantum dot-conjugated secondary antibodies to enhance visualization of low-abundance LRRC3B. Additionally, consider using positive control cells with LRRC3B overexpression to validate antibody functionality and experimental conditions before proceeding with cancer cell lines. When designing RT-qPCR experiments, utilize high-efficiency primers spanning exon-exon junctions, implement pre-amplification steps for low-abundance transcripts, and consider digital PCR platforms that offer superior sensitivity for rare transcript detection.

How can researchers optimize LRRC3B antibody-based immunohistochemistry for analyzing clinical samples?

Optimizing LRRC3B antibody-based immunohistochemistry for clinical samples requires careful attention to several critical parameters. Begin with proper tissue fixation—10% neutral-buffered formalin fixation for 24-48 hours provides optimal preservation of LRRC3B epitopes while maintaining tissue architecture. For antigen retrieval, heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be empirically tested to determine which better exposes LRRC3B epitopes without causing tissue degradation.

Antibody dilution optimization is essential—begin with manufacturer's recommended dilution and test a range (e.g., 1:50 to 1:500) to identify the optimal signal-to-noise ratio. When selecting primary antibodies, prioritize those validated specifically for immunohistochemistry applications, as antibodies optimized solely for Western blot may not perform consistently on tissue sections . Overnight incubation at 4°C typically yields superior results compared to shorter incubations at higher temperatures.

For signal development, polymer-based detection systems offer greater sensitivity than standard avidin-biotin methods, particularly useful for tissues with expected low LRRC3B expression. Include positive control tissues (testis, skeletal muscle, cerebral cortex) and negative controls (primary antibody omission) in each staining batch . For multiplex studies investigating LRRC3B expression alongside immune markers, sequential staining protocols with appropriate antibody stripping between rounds provides cleaner results than simultaneous multiplexing. Finally, standardize scoring systems (H-score or Allred) for consistent quantification across samples, preferably with blinded evaluation by multiple pathologists.