RPLP2 Antibody

What is RPLP2 and what are its primary cellular functions?

RPLP2 (ribosomal protein, large, P2) is an integral component of the ribosomal stalk structure that constitutes the ribosomal P complex along with RPLP0 and RPLP1. This 115 amino acid protein has a calculated molecular weight of approximately 12 kDa . RPLP2 plays a crucial role in protein synthesis by facilitating the recruitment of translational factors to ribosomes, thereby enabling efficient translation of mRNAs . The P complex formed by these proteins is essential for normal cellular protein synthesis and metabolism. Research has demonstrated that RPLP2 is evolutionarily conserved across species, with significant homology observed between human and mosquito (Aedes aegypti) RPLP2 genes (69.37% nucleotide sequence identity) .

What are the validated applications for RPLP2 antibodies in research?

RPLP2 antibodies have been validated for several critical research applications, with Western blotting (WB) and immunohistochemistry (IHC) being the most commonly employed techniques. For Western blot applications, the recommended dilution range is 1:500-1:2000, while for IHC applications, dilutions of 1:50-1:500 are typically used . Published literature confirms successful application of RPLP2 antibodies in these contexts with multiple species reactivity, including human and mouse samples. Positive Western blot detection has been documented in HL-60 cells and mouse liver tissue, while positive IHC staining has been observed in mouse kidney tissue and human breast cancer tissue . The versatility of RPLP2 antibodies makes them valuable tools for studying this protein's expression and localization across different experimental models.

How should researchers optimize antibody conditions for RPLP2 detection?

For optimal RPLP2 detection, researchers should conduct preliminary titration experiments to determine the ideal antibody concentration for their specific sample types and detection methods. When performing IHC, antigen retrieval with TE buffer at pH 9.0 is recommended, although citrate buffer at pH 6.0 can serve as an alternative . For Western blot applications, optimization of blocking conditions, incubation times, and washing steps is essential for specific signal detection. Given that reactivity can vary between species and tissue types, validation using appropriate positive controls (such as HL-60 cells for human studies or mouse liver tissue) is highly recommended before conducting comprehensive experiments. Researchers should be aware that sample-dependent variations may necessitate additional optimization beyond the manufacturer's recommended protocols .

How is RPLP2 implicated in cancer biology, particularly hepatocellular carcinoma?

RPLP2 has emerged as a significant factor in cancer progression, particularly in hepatocellular carcinoma (HCC). Bioinformatic analyses using TCGA, GTEx, HCCDB, and other databases have revealed that RPLP2 is significantly overexpressed in HCC compared to normal liver tissue . This elevated expression strongly correlates with advanced clinicopathological features and predicts poor prognosis in HCC patients. The diagnostic value of RPLP2 is particularly notable, with ROC curve analysis demonstrating excellent performance (AUC = 0.906) in distinguishing HCC tumors from normal controls . Mechanistically, RPLP2 functions as an inhibitor of ferroptosis by positively associating with the ferroptosis suppressor GPX4, thereby promoting tumor progression. Experimental validation through functional assays (CCK8, transwell, and colony formation) and xenograft models confirms that RPLP2 knockdown significantly suppresses HCC cell proliferation, migration, and tumor growth both in vitro and in vivo .

What is the role of RPLP2 in viral infection mechanisms?

RPLP2, along with RPLP1, has been identified as an essential host factor for flavivirus infections. Research using RNA interference approaches demonstrated that knockdown of RPLP1/2 in A549 cells reduced infectious virus production by up to 2 orders of magnitude for dengue virus (DENV) and yellow fever virus (YFV) . Similar reductions were observed in HuH-7 cells (15- to 30-fold for DENV and 10- to 50-fold for YFV). The mechanism appears to involve early viral protein accumulation, as experiments with a DENV-2 luciferase reporter virus showed significantly reduced luciferase levels at 4 hours post-infection in RPLP1/2-depleted cells, even in the presence of the NS5 inhibitor NITD008 . This suggests that RPLP1/2 affects an early stage of the viral life cycle, likely viral translation, rather than RNA replication. The conservation of RPLP1/2 genes between humans and mosquito vectors further suggests their importance in viral transmission cycles.

How does RPLP2 regulation impact immune cell infiltration in tumors?

RPLP2 expression significantly correlates with immune cell infiltration patterns in tumors, particularly in HCC. Single-sample gene set enrichment analysis (ssGSEA) has revealed that RPLP2 expression positively correlates with the infiltration of NK CD56 bright cells and Th2 cells, while negatively correlating with central memory T cells (Tcm) . These findings have been validated through immunohistochemistry, confirming that high RPLP2 expression is associated with increased NK CD56 bright and Th2 cell infiltration and decreased Tcm cell infiltration in HCC tissues . This immunomodulatory effect of RPLP2 may contribute to its impact on tumor progression and patient outcomes. The mechanisms by which RPLP2 affects immune cell recruitment and function remain an active area of investigation, but these findings suggest that RPLP2-targeted therapies might influence the tumor immune microenvironment in addition to their direct effects on cancer cells.

What methodologies are recommended for studying RPLP2 knockdown effects?

For effective RPLP2 knockdown studies, researchers should consider using small interfering RNA (siRNA) approaches with carefully designed and validated siRNA sequences. Based on published protocols, a pool of siRNAs targeting RPLP1/2 at a total concentration of 30 nM has been successfully employed in cell culture models . Following transfection, a 48-hour incubation period is typically sufficient to achieve effective knockdown before proceeding with functional assays. For protein synthesis studies, metabolic labeling with 35S methionine/cysteine (0.1 mCi for 30 minutes) after depleting intracellular methionine and cysteine pools (20-minute incubation in depleted media) can provide quantitative data on translation effects . Downstream analysis should include verification of knockdown efficiency through Western blotting or qRT-PCR, followed by appropriate functional assays such as cell proliferation, migration, or viral infection studies depending on the research question.

How can researchers investigate the role of RPLP2 in cellular stress responses?

Studies examining RPLP2's role in cellular stress responses should incorporate multiple complementary approaches. Evidence indicates that disruption of the ribosomal P complex through RPLP2 knockdown induces reactive oxygen species (ROS) accumulation, triggering endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) . To investigate this pathway, researchers should measure ROS levels using fluorescent probes and assess activation of the MAPK1/ERK2 signaling pathway through phosphorylation-specific antibodies. For UPR activation, examination of the EIF2AK3/PERK-EIF2S1/eIF2α-EIF2S2-EIF2S3-ATF4/ATF-4 and ATF6/ATF-6-dependent arms is recommended through Western blotting for these markers . To establish causality, antioxidant treatments can be employed to determine if preventing ROS accumulation blocks subsequent stress responses. Additionally, autophagy markers (such as LC3-II conversion) should be monitored, as RPLP2 deficiency has been shown to induce autophagy as a survival mechanism. For comprehensive analysis, these studies should be combined with cell viability assays in the presence of autophagy inhibitors to assess whether autophagy inhibition redirects cells toward apoptotic death .

What techniques are optimal for analyzing RPLP2's impact on ferroptosis in cancer models?

To investigate RPLP2's role in ferroptosis regulation, researchers should implement a multi-faceted approach combining molecular, cellular, and biochemical techniques. Western blot analysis should be employed to examine the relationship between RPLP2 and ferroptosis regulators, particularly GPX4, with which RPLP2 has been shown to positively associate . Cell viability assays (such as CCK8) should be conducted under conditions that induce ferroptosis, comparing control and RPLP2-depleted cells. Key ferroptosis indicators including glutathione (GSH) levels and lipid reactive oxygen species (ROS) should be quantified using specific detection assays . For in vivo validation, xenograft models with RPLP2 knockdown or overexpression, followed by immunohistochemical analysis of ferroptosis markers in tumor tissues, provide valuable translational insights. Rescue experiments, where ferroptosis inhibitors are added to RPLP2-depleted cells, can establish causal relationships. Finally, transcriptomic analysis comparing control and RPLP2-depleted cells can identify broader transcriptional networks affected by RPLP2 manipulation that may contribute to ferroptosis regulation.

How can RPLP2 expression analysis be incorporated into cancer prognosis models?

RPLP2 expression analysis can be effectively integrated into cancer prognostic models through a systematic approach. Research has demonstrated that RPLP2 expression levels have significant prognostic value, particularly in HCC. Nomograms combining RPLP2 expression with critical clinical features have been developed that exhibit high predictive accuracy for 1-, 3-, and 5-year survival probabilities in HCC patients . For researchers developing similar prognostic models, RNA-seq data from tumor samples should be normalized using established protocols, and RPLP2 expression thresholds for risk stratification should be determined through ROC curve analysis. Time-dependent ROC curves can assess prediction accuracy for different survival timeframes, with published data showing AUC values of 0.589, 0.584, and 0.591 for 1-, 3-, and 5-year survival rates respectively in HCC . Integration with clinical parameters (tumor stage, grade, vascular invasion status) through multivariate Cox regression analysis can generate comprehensive nomograms with improved predictive power. Validation across independent cohorts is essential to establish the robustness and generalizability of such prognostic models.

What are the potential therapeutic implications of targeting RPLP2 in cancer?

The emerging role of RPLP2 in cancer progression, particularly through ferroptosis inhibition, presents promising therapeutic opportunities. Drug sensitivity analysis has identified several compounds with potential efficacy against RPLP2-overexpressing tumors, including methylundecylpiperidine, trans, iyomycin b1, and destruxin b . Additionally, drugs targeting DNA methylation of RPLP2 (such as tp4ek-k6, Ibrutinib, and indole-2,3-dione,3-[(o-chlorophenyl)hydrazone]) have shown significant sensitivity in cases with elevated RPLP2 methylation levels . Development of therapeutic strategies should focus on either direct inhibition of RPLP2 function or promotion of ferroptosis to counteract RPLP2's protective effect in cancer cells. Combination approaches targeting both RPLP2 and its downstream effector GPX4 might provide synergistic benefits. For preclinical evaluation of such therapies, researchers should utilize established cancer cell lines with validated RPLP2 expression patterns, xenograft models, and patient-derived organoids to assess efficacy and specificity. Monitoring ferroptosis markers and immune cell infiltration patterns would provide valuable insights into the mechanisms and broader effects of RPLP2-targeted therapies.

How might RPLP2 antibodies be utilized in developing diagnostic tools for cancer detection?

RPLP2 antibodies hold significant potential for developing cancer diagnostic tools based on the protein's differential expression patterns in various malignancies. The high diagnostic value of RPLP2 in distinguishing HCC from normal tissue (AUC = 0.906) suggests its utility as a biomarker . For developing immunohistochemistry-based diagnostic assays, researchers should optimize RPLP2 antibody protocols with appropriate antigen retrieval methods (TE buffer at pH 9.0 or citrate buffer at pH 6.0) . Digital pathology approaches incorporating RPLP2 immunostaining intensity quantification could improve diagnostic objectivity. For liquid biopsy applications, sensitive detection methods for circulating RPLP2 protein or autoantibodies against RPLP2 in patient blood samples should be explored. Multiplexed immunoassays combining RPLP2 with established cancer markers might enhance diagnostic accuracy. Development of such tools should include comprehensive validation across diverse patient cohorts with varying cancer stages, and establishment of standardized scoring systems with clinically relevant cutoff values. The excellent diagnostic performance of RPLP2 in multiple HCC subgroups (including different grades, T stages, and clinical stages) suggests broad applicability of RPLP2-based diagnostic approaches .

What controls should be included when using RPLP2 antibodies for experimental validation?

Proper experimental controls are crucial when using RPLP2 antibodies to ensure validity and reproducibility of results. For Western blot applications, positive controls should include samples known to express RPLP2, such as HL-60 cells for human studies or mouse liver tissue for murine experiments . Negative controls should incorporate RPLP2 knockdown samples (siRNA or shRNA treated) to confirm antibody specificity. For immunohistochemistry, positive control tissues such as mouse kidney or human breast cancer tissue should be included in each staining run . Additionally, primary antibody omission controls and isotype-matched irrelevant antibody controls are essential to assess non-specific binding. When investigating RPLP2 function through knockdown experiments, multiple siRNA sequences targeting different regions of RPLP2 mRNA should be employed to rule out off-target effects. Rescue experiments, where RPLP2 expression is restored through an siRNA-resistant construct, provide powerful validation of phenotypic effects. For all quantitative analyses, technical and biological replicates (minimum n=3) are necessary, with appropriate statistical methods applied to determine significance of findings.

How can researchers effectively analyze RPLP2's role in translation through ribosome profiling?

Ribosome profiling offers powerful insights into RPLP2's role in translation regulation. When designing such experiments, researchers should consider the approach used in search result , where metagene analysis was restricted to genes with at least one dataset containing 64 mapped reads, while ignoring genes with zero reads unless their mean across all datasets exceeded 64. For codon-specific analyses, footprints of 29-35 nucleotides should be selected, with A-site mapping using specific offsets (+15 for footprints of lengths 29-30, +16 for lengths 31-33, and +17 for lengths 34-35) . Differential expression analysis between control and RPLP2-depleted conditions should be performed using reliable statistical packages such as DESeq2, both at the transcriptional and translational levels separately. For metagene analysis, normalized reads of 10-50 bp should be aligned by their 5' end as previously described in the literature . This comprehensive approach allows researchers to distinguish between transcriptional and translational effects of RPLP2 depletion, potentially identifying specific mRNAs or codons that are particularly dependent on RPLP2 function for efficient translation.

What methodological approaches are recommended for studying RPLP2 in viral infection models?

For investigating RPLP2's role in viral infection, researchers should implement a systematic approach beginning with effective RPLP2 depletion. RNA interference through pooled siRNAs at approximately 30 nM has proven effective in cell culture models . Following knockdown verification, cells should be infected with the virus of interest at a controlled multiplicity of infection (MOI), with MOI=1 being suitable for many experimental designs . Both viral replication and infectivity should be assessed through complementary methods: (1) immunofluorescence for viral antigens to determine infection rates, (2) quantification of infectious virus in cell supernatants through plaque assays or TCID50 assays, and (3) measurement of viral RNA levels through qRT-PCR. To investigate mechanisms, reporter viruses (such as luciferase-expressing constructs) enable quantitative assessment of viral protein accumulation at early time points (e.g., 4 hours post-infection) . Inclusion of specific viral replication inhibitors (such as the NS5 inhibitor NITD008 for flaviviruses) can help distinguish between effects on translation versus replication . For in vivo relevance, vector models such as mosquito infection studies with RPLP1/2 knockdown through dsRNA injection provide valuable insights into the role of these proteins in viral transmission cycles.