RPRD1A Antibody

What is RPRD1A and why is it important in research?

RPRD1A (Regulation of Nuclear Pre-mRNA Domain-containing protein 1A) is a protein that plays significant roles in transcriptional regulation and oxidative stress response in cells. Research has demonstrated that RPRD1A is particularly important due to its upregulation in hepatocellular carcinoma (HCC) tissues compared to corresponding normal liver tissues . RPRD1A contains a CID (CTD-Interacting Domain) and coiled-coil domains that facilitate its interaction with RNA polymerase II and other transcriptional regulators . The significance of RPRD1A in cancer research lies in its correlation with aggressive tumor features, including larger tumor size, vein invasion, and advanced TNM and BCLC staging in HCC patients . Additionally, RPRD1A forms complexes with its paralog RPRD1B (80% similarity) and participates in transcription control mechanisms that regulate gene expression .

How do I select the appropriate RPRD1A antibody for Western blotting?

When selecting an RPRD1A antibody for Western blotting, consider these methodological aspects:

• Epitope recognition: Choose antibodies that target well-conserved regions of RPRD1A that won't be affected by potential post-translational modifications in your experimental conditions.

• Validation status: Look for antibodies that have been validated specifically for Western blotting in tissues or cell lines similar to your experimental model. In published research, RPRD1A has been successfully detected in HCC tissues and cell lines including MHCCLM3 and Huh7 .

• Species reactivity: Ensure the antibody is reactive to your species of interest. The research data available primarily shows RPRD1A detection in human samples .

• Expected molecular weight: RPRD1A should appear at the appropriate molecular weight on your blot (usually in the 35-40 kDa range, but verify the exact size for your experimental system).

• Control samples: Include appropriate positive controls such as MHCCLM3 cells, which have been documented to express RPRD1A at detectable levels .

What are the recommended protocols for immunohistochemical detection of RPRD1A?

For immunohistochemical (IHC) detection of RPRD1A, researchers should follow these methodological guidelines:

• Tissue preparation: Use formalin-fixed, paraffin-embedded (FFPE) tissue sections, as these have been successfully used in published RPRD1A studies. For example, researchers have examined RPRD1A expression in cohorts of 267 HCC cases using IHC .

• Antigen retrieval: Apply appropriate antigen retrieval methods, typically heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), to expose the RPRD1A epitopes that may be masked during fixation.

• Scoring system: Implement a standardized scoring system similar to that used in published research. For instance, RPRD1A expression has been scored on a scale of 0-3 based on staining intensity, with scores 0-1 classified as low expression and scores 2-3 as high expression .

• Controls: Include appropriate positive controls (such as HCC tissues known to express RPRD1A) and negative controls (normal liver tissues or antibody diluent alone) to validate staining specificity.

• Results interpretation: When analyzing results, remember that in HCC studies, approximately 34.8% of tumor tissues showed high RPRD1A expression compared to 21.4% of para-tumor tissues, indicating differential expression patterns between normal and cancer tissues .

How can I use RPRD1A antibodies to investigate its role in oxidative stress response?

RPRD1A plays a critical role in cellular antioxidant capacity and oxidative stress responses. To investigate this function:

• Co-immunoprecipitation (Co-IP) assays: Use anti-RPRD1A antibodies to pull down RPRD1A and its interacting partners under different oxidative conditions. Research has shown that RPRD1A inhibits the association between Keap1 and NRF2 . Design your Co-IP experiments to detect these interactions.

• ROS measurement: Combine RPRD1A knockdown or overexpression with ROS detection assays. Studies indicate that RPRD1A depletion significantly increases ROS production in HCC cells, with even greater accumulation when treated with H₂O₂ .

• Measure antioxidant capacity: Assess the GSH/GSSG ratio in cells with manipulated RPRD1A levels. Research has demonstrated that RPRD1A deletion induces a strong decrease in the GSH/GSSG ratio, indicating impaired antioxidant capacity .

• Investigate NRF2 stabilization: Use Western blotting to examine how RPRD1A affects NRF2 protein accumulation under oxidative stress conditions. RPRD1A knockdown has been shown to decrease NRF2 accumulation under H₂O₂ treatment .

• Antioxidant enzyme expression: Measure the expression of antioxidant enzymes regulated by ARE elements using qPCR and Western blotting. RPRD1A positively regulates the mRNA levels of several antioxidant enzymes including SOD1, SOD2, GCLC, GCLM, ANT, NQO1, HO1, PRDX4, and PRDX6 .

What approaches can I use to study RPRD1A interactions with the ubiquitin-proteasome system?

RPRD1A has been shown to affect protein ubiquitination and degradation pathways. To investigate these interactions:

• Ubiquitination assays: Perform ubiquitination assays by co-transfecting cells with HA-ubiquitin and manipulating RPRD1A expression. Research has shown that RPRD1A decreases the ubiquitination of p62 .

• Proteasome inhibition studies: Combine RPRD1A manipulation with proteasome inhibitors (e.g., MG132) to assess how RPRD1A affects protein stability through proteasomal degradation. Published work has demonstrated that RPRD1A inhibits ubiquitin-proteasome mediated degradation of NRF2 .

• Mass spectrometry analysis: Use immunoprecipitation with RPRD1A antibodies followed by mass spectrometry to identify novel interacting partners involved in ubiquitination pathways. This approach identified TRIM21, an E3 ubiquitin ligase, as an interactor with RPRD1A .

• Half-life determination: Measure the half-life of NRF2 or other target proteins in cells with different RPRD1A expression levels using cycloheximide chase assays. Research has shown that RPRD1A overexpression prolongs the half-life of NRF2 .

• Co-IP confirmation: Validate interactions between RPRD1A and ubiquitin pathway components like TRIM21 using targeted Co-IP experiments. Endogenous interaction between RPRD1A and TRIM21 has been confirmed through this approach .

How do I design experiments to investigate RPRD1A's role in transcriptional regulation?

RPRD1A participates in complex transcription control mechanisms. To study these functions:

• RNA metabolic labeling: Use 4sU labeling followed by RNA sequencing to assess how RPRD1A affects nascent RNA synthesis. This approach revealed that RPRD1A depletion affects RNA synthesis, though less dramatically than RPRD2 or RPRD1B depletion .

• Luciferase reporter assays: Employ dual-luciferase reporter gene assays to examine RPRD1A's influence on specific promoter elements. For example, ARE-luciferase activity decreased under oxidative stress when RPRD1A was knocked down .

• Chromatin immunoprecipitation (ChIP): Use ChIP assays with RPRD1A antibodies to identify genomic regions where RPRD1A binds, potentially in complex with RNA polymerase II.

• RNA polymerase II phosphorylation analysis: Examine how RPRD1A affects the phosphorylation state of RNA polymerase II CTD, particularly at S5, as RPRD1A has been shown to bind to the CTD phosphorylated at this position .

• Transcriptome analysis: Compare the effects of RPRD1A knockdown or overexpression on the transcriptome. Research has shown that RPRD1A knockdown resulted in the altered expression of 3693 upregulated and 2589 downregulated transcription units, with a ratio of 1.4 .

What are the suitable controls and validation approaches for RPRD1A knockdown or overexpression experiments?

When designing RPRD1A manipulation experiments:

• Knockdown validation: Confirm RPRD1A knockdown efficiency at both mRNA and protein levels. Published studies typically verify knockdown using both real-time PCR and Western blotting .

• Rescue experiments: Perform rescue experiments by re-expressing RPRD1A in knockdown cells to confirm that observed phenotypes are specifically due to RPRD1A loss. For example, cell death caused by RPRD1A knockdown under H₂O₂ treatment could be rescued by antioxidants like N-acetyl-L-cysteine (NAC) .

• Multiple knockdown strategies: Use at least two independent shRNA sequences targeting RPRD1A to rule out off-target effects. Published research has successfully employed this approach .

• Paralog-specific controls: Since RPRD1A has paralogs (RPRD1B and RPRD2), include controls that knock down or overexpress these proteins to distinguish paralog-specific functions from redundant ones .

• Physiological expression levels: When overexpressing RPRD1A, include controls to verify that expression levels are within physiologically relevant ranges observed in tissues of interest.

How can I investigate the clinical significance of RPRD1A expression in cancer samples?

To study RPRD1A's relevance in cancer:

How can I address non-specific binding issues with RPRD1A antibodies?

When encountering non-specific binding:

• Antibody validation: Verify antibody specificity using positive controls (cells overexpressing RPRD1A) and negative controls (RPRD1A knockdown cells).

• Blocking optimization: Test different blocking reagents (BSA, milk, commercial blockers) and concentrations to minimize background signals.

• Antibody dilution: Titrate antibody concentrations to find the optimal dilution that maximizes specific signal while minimizing background.

• Buffer composition: Adjust buffer composition (salt concentration, detergent type and concentration) to enhance specificity.

• Paralog cross-reactivity: Given the 80% similarity between RPRD1A and RPRD1B , confirm that your antibody doesn't cross-react with RPRD1B by testing in cells where RPRD1B is specifically knocked down.

What are the common pitfalls when comparing RPRD1A expression across different experimental conditions?

When comparing RPRD1A expression:

• Reference gene selection: Choose appropriate reference genes for normalization in qPCR experiments, as RPRD1A is involved in transcriptional regulation and may indirectly affect common housekeeping genes.

• Loading control selection: For Western blotting, select loading controls that are stable across your experimental conditions and are not affected by RPRD1A manipulation.

• Expression heterogeneity: Consider the heterogeneity of RPRD1A expression within tissues. In HCC, about 70% of PVTT presented strong expression of RPRD1A , indicating potential variability.

• Subcellular localization: Account for potential changes in RPRD1A subcellular localization under different experimental conditions, as this may affect antibody accessibility and signal intensity.

• Post-translational modifications: Be aware that post-translational modifications might affect antibody binding, potentially leading to false-negative results under conditions that promote such modifications.

How is RPRD1A involved in cancer cell response to oxidative stress?

Recent research has revealed that RPRD1A plays a crucial role in cancer cell adaptation to oxidative stress:

• NRF2 stabilization: RPRD1A enhances the nuclear translocation of NRF2, a key transcription factor that induces gene expression to counteract oxidative stress .

• Antioxidant enzyme regulation: RPRD1A positively regulates the expression of numerous antioxidant enzymes, including SOD1, SOD2, GCLC, GCLM, NQO1, and HO1 .

• GSH/GSSG ratio maintenance: RPRD1A helps maintain cellular redox homeostasis by increasing the GSH/GSSG ratio, enhancing cellular antioxidant capacity .

• Keap1-NRF2 pathway modulation: RPRD1A inhibits the association between Keap1 and NRF2, thereby preventing NRF2 degradation and promoting antioxidant responses .

• TRIM21-p62 interaction: RPRD1A interacts with the E3 ubiquitin ligase TRIM21 and decreases the ubiquitination of p62, which affects p62-dependent sequestration of Keap1 .

What is the relationship between RPRD1A and its paralogs in transcriptional regulation?

The relationship between RPRD1A and its paralogs (RPRD1B and RPRD2) in transcriptional regulation is complex:

• Complex formation: RPRD1B forms mutually exclusive complexes with RPRD1A and RPRD2, potentially coordinating their roles in transcription control .

• Differential effects on transcription: While all three RPRDs affect RNA synthesis, RPRD2 has the most pronounced effect, followed by RPRD1B, with RPRD1A showing the least impact .

• Transcriptional outcomes: RPRD1A knockdown affects fewer transcription units than RPRD1B or RPRD2 knockdown, with a lower ratio of upregulated to downregulated genes (1.4 for RPRD1A compared to 2.5 for RPRD1B and 4.1 for RPRD2) .

• RNA polymerase II interaction: All three RPRDs can interact with RNA polymerase II CTD, but may have different effects on CTD modification and downstream transcription events .

• Shared structural features: RPRD1A and RPRD1B share 80% similarity and both contain CID and coiled-coil domains that mediate their interaction and binding to the CTD phosphorylated at S5 .

What are promising therapeutic approaches targeting RPRD1A in cancer?

Based on current knowledge, several therapeutic approaches targeting RPRD1A show promise:

• Oxidative stress sensitization: Genetic knockdown of RPRD1A sensitizes cancer cells to platinum-induced cell death by disturbing redox homeostasis , suggesting a potential approach to enhance chemotherapy efficacy.

• Combination therapies: Combining RPRD1A inhibition with agents that increase ROS production could create synthetic lethality in cancer cells, as RPRD1A depletion impairs the cellular response to oxidative stress .

• NRF2 pathway targeting: Since RPRD1A stabilizes NRF2, combining RPRD1A inhibition with other approaches that target the NRF2 pathway might provide synergistic effects in cancer treatment.

• Transcription regulation: Given RPRD1A's role in transcriptional control , targeting its interaction with transcriptional machinery might affect cancer-specific gene expression programs.

• Metastasis prevention: As RPRD1A is highly expressed in metastatic tissues like PVTT , targeting RPRD1A might help prevent cancer metastasis, a major cause of cancer mortality.

How might emerging technologies advance our understanding of RPRD1A function?

Emerging technologies offer new opportunities to elucidate RPRD1A functions:

• Single-cell analysis: Single-cell RNA sequencing and proteomics could reveal cell-specific roles of RPRD1A in heterogeneous tissues and tumors.

• CRISPR screens: Genome-wide CRISPR screens could identify synthetic lethal interactions with RPRD1A in different cancer contexts.

• Structural biology approaches: Cryo-electron microscopy and other structural biology techniques might elucidate how RPRD1A interactions with its binding partners affect protein function and stability.

• Live-cell imaging: Advanced imaging techniques could monitor RPRD1A dynamics, nuclear translocation, and co-localization with binding partners in real-time.

• Proteomics: Large-scale proteomics approaches could comprehensively map the RPRD1A interactome under various cellular conditions, expanding on the initial findings that identified TRIM21 as an RPRD1A-interacting protein .