NUPR1 Antibody

What is NUPR1 and why is it significant in research?

NUPR1 is a small nuclear protein (approximately 8.9 kilodaltons) that functions as a transcriptional regulator . Initially discovered in 1997 during studies on pancreatitis and pancreatic development, NUPR1 has since been firmly established as playing crucial roles in cancer development and progression, as well as in various other pathological conditions . The protein is also known by several alternative names including p8, COM1 (candidate of metastasis 1), and is recognized as a transcriptional regulator . NUPR1's significance in research stems from its involvement in critical cellular processes including cell death mechanisms, gene transcription regulation, and most notably, its elevated expression in metastatic tumors compared to primary tumors .

What types of NUPR1 antibodies are available for research applications?

NUPR1 antibodies are available in multiple formats to accommodate diverse research needs. Based on the available information, researchers can access polyclonal and monoclonal antibodies against NUPR1, with various host species including rabbit, goat, and mouse-derived options . These antibodies come in different conjugation states including unconjugated formats and biotin-conjugated variants for specialized detection methods . For application-specific requirements, researchers can select antibodies validated for Western blotting, ELISA, immunofluorescence (IF), immunohistochemistry (IHC) on both frozen and paraffin-embedded sections, and immunocytochemistry (ICC) . Antibodies targeting specific regions of NUPR1, such as the C-terminal region or amino acids 31-82, are also available for more targeted experimental approaches .

How do researchers validate the specificity of NUPR1 antibodies?

Validating antibody specificity is critical for ensuring experimental reliability. For NUPR1 antibodies, validation typically involves a multi-tiered approach. Initially, researchers should perform Western blot analysis using both positive control samples (tissues or cell lines known to express NUPR1, such as gastric cancer cells) and negative controls where NUPR1 expression has been knocked down using shRNA or similar techniques . The appearance of a single band at approximately 8.9 kDa indicates specificity. Cross-reactivity testing across multiple species is essential since NUPR1 antibodies may have variable reactivity across human, mouse, rat, and other species depending on the specific product .

For immunohistochemistry applications, researchers should compare staining patterns in tissues with known NUPR1 expression levels and perform parallel experiments with NUPR1-depleted samples. In immunofluorescence studies, co-localization with other nuclear markers helps confirm the expected nuclear localization pattern of NUPR1. When interpreting validation results, researchers should consider that NUPR1 expression can be altered by experimental conditions, particularly in response to anti-cancer drugs like doxorubicin, 5-Fluorouracil, and oxaliplatin, which have been shown to upregulate NUPR1 expression .

How does NUPR1 expression correlate with cancer progression?

The upregulation pattern suggests NUPR1 may serve as a potential biomarker for cancer progression. For researchers investigating NUPR1 as a biomarker, it is important to note that expression analysis requires careful quantification through multiple methodologies including qPCR, immunoblotting, and immunohistochemistry to establish reliable expression profiles across different cancer types and stages .

What cellular mechanisms are regulated by NUPR1 in cancer cells?

NUPR1 influences multiple cellular mechanisms critical to cancer progression. In gastric cancer models, NUPR1 knockdown experiments have demonstrated its significant regulation of several key processes:

• Cell Migration and Invasion: NUPR1 depletion inhibits migration capabilities as demonstrated in transwell and wound closure assays, suggesting its role in promoting metastatic potential .

• Apoptosis Regulation: NUPR1 knockdown enhances apoptotic populations as detected by flow cytometry, with corresponding molecular changes including upregulation of cleaved Caspase-3 and downregulation of Bcl-2 .

• Epithelial-Mesenchymal Transition (EMT): NUPR1 regulates the EMT process, with its depletion increasing E-cadherin (epithelial marker) and reducing N-cadherin, Vimentin, and Snail (mesenchymal markers) .

• Drug Resistance: NUPR1 plays a significant role in resistance to anti-cancer drugs. Studies show that treatment with 5-Fluorouracil, oxaliplatin, and doxorubicin upregulates NUPR1 expression in cancer cells, and NUPR1 depletion increases sensitivity to these treatments .

These mechanisms position NUPR1 as a potential therapeutic target in cancer research, particularly in addressing drug resistance mechanisms.

What signaling pathways interact with NUPR1 in tumor progression?

NUPR1 interacts with several critical signaling pathways in cancer cells, forming a complex regulatory network. The Yes-associated protein (YAP) pathway has been identified as a key interactor with NUPR1, particularly in the context of drug resistance in gastric cancer . Research has demonstrated that YAP activation is upregulated in doxorubicin-resistant gastric cancer cells and can regulate NUPR1 expression to affect drug resistance .

The AKT pathway serves as another important signaling mechanism connected to NUPR1 function. Studies have shown that NUPR1 overexpression can reverse the effects of YAP knockdown on cell malignancy and drug resistance through regulation of the AKT pathway . Additionally, NUPR1 influences p21 activation, which plays a crucial role in cell cycle regulation and apoptosis .

The following signaling network has been demonstrated in experimental models:

• YAP activation → NUPR1 upregulation → AKT activation → Enhanced drug resistance

• NUPR1 overexpression can rescue the phenotypes caused by YAP ablation

• AKT inhibition can reverse the effects of NUPR1 overexpression

Researchers investigating these pathways should consider the interdependence of these signaling components when designing experiments targeting NUPR1 in cancer models.

What are the optimal conditions for using NUPR1 antibodies in Western blotting?

When performing Western blotting with NUPR1 antibodies, researchers should optimize several key parameters for successful detection of this small (8.9 kDa) protein. For sample preparation, using RIPA buffer supplemented with protease inhibitors is recommended to prevent degradation of NUPR1. Due to the small size of NUPR1, high-percentage (12-15%) SDS-PAGE gels provide better resolution for accurate detection .

For blocking, 5% non-fat milk or BSA in TBST is typically effective, though some antibodies may have specific recommendations. Primary NUPR1 antibody dilutions generally range from 1:500 to 1:2000 depending on the specific product, with overnight incubation at 4°C typically yielding the best results . When detecting NUPR1 after drug treatments (such as doxorubicin, 5-Fluorouracil, or oxaliplatin), researchers should be aware that these treatments can increase NUPR1 expression levels and may require adjustment of antibody concentrations .

Using appropriate positive controls (such as MKN45 or AGS gastric cancer cell lines) is crucial, as is the inclusion of NUPR1-knockdown samples as negative controls to confirm antibody specificity . For data analysis, normalization to appropriate loading controls is essential, with special attention to the low molecular weight range where NUPR1 appears.

How should NUPR1 antibodies be used in immunohistochemistry studies?

For immunohistochemistry (IHC) applications with NUPR1 antibodies, both frozen and paraffin-embedded tissue sections can be used, though specific protocol optimizations may be required for each format . Antigen retrieval is particularly important for paraffin-embedded tissues, with citrate buffer (pH 6.0) commonly used, though specific products may have alternative recommendations.

Based on published research, NUPR1 shows predominantly nuclear localization, consistent with its function as a transcriptional regulator. When evaluating IHC results, researchers should assess both staining intensity and the percentage of positive cells to generate a comprehensive expression score . For cancer tissues, comparing NUPR1 expression between tumor and adjacent normal tissues provides valuable insight, as increased expression has been observed in cancerous tissues .

When conducting IHC studies on clinical samples, researchers should correlate NUPR1 expression with clinical parameters including tumor size, tumor stage, and lymph node metastasis, as these have shown significant correlations in previous studies . For multiplex staining approaches, combining NUPR1 detection with markers of epithelial-mesenchymal transition (such as E-cadherin and N-cadherin) can provide additional insights into its role in cancer progression .

What controls are essential when using NUPR1 antibodies in flow cytometry?

Flow cytometry applications using NUPR1 antibodies require careful control selection to ensure reliable data interpretation. For intracellular staining of NUPR1, proper fixation and permeabilization protocols are essential since NUPR1 is primarily a nuclear protein. Researchers should include isotype controls matched to the NUPR1 antibody's host species and immunoglobulin class to account for non-specific binding .

Biological controls should include both positive control samples (cell lines with confirmed NUPR1 expression such as MKN45 or AGS gastric cancer cells) and negative controls such as NUPR1-knockdown cells generated through shRNA or CRISPR-Cas9 approaches . When studying apoptosis in relation to NUPR1, as shown in previous research, concurrent staining with Annexin V-FITC and PI alongside NUPR1 detection provides valuable information about the relationship between NUPR1 expression and apoptotic status .

For drug response studies, researchers should include time-course analyses (0h, 24h, 48h) after treatment with relevant compounds such as doxorubicin, as NUPR1 expression has been shown to increase in a time-dependent manner following such treatments . This temporal information helps establish the dynamic relationship between drug exposure and NUPR1 expression changes.

How can researchers effectively study NUPR1's role in drug resistance mechanisms?

To investigate NUPR1's involvement in drug resistance, researchers should implement a comprehensive experimental approach. Initial characterization should establish baseline drug sensitivity profiles using MTT or similar viability assays with escalating concentrations of relevant therapeutics (such as doxorubicin, 5-Fluorouracil, and oxaliplatin) in the cancer cell lines of interest . This provides IC50 values that serve as reference points for subsequent experiments.

For mechanistic studies, researchers should establish NUPR1-knockdown models using validated shRNA constructs, with knockdown efficiency verified at both mRNA and protein levels using qPCR and Western blotting respectively . Drug sensitivity assays should then be repeated in both control and NUPR1-depleted cells to quantify changes in IC50 values and establish NUPR1's contribution to drug resistance .

To understand the temporal dynamics of NUPR1 in drug response, time-course analyses measuring NUPR1 expression following drug exposure at multiple timepoints (0h, 24h, 48h) provides insight into the kinetics of NUPR1 upregulation . This helps determine whether NUPR1 induction is an early or late response to therapeutic stress.

For pathway analyses, researchers should examine the interplay between NUPR1 and other signaling components, particularly the YAP-AKT-p21 axis. This can be accomplished through:

• Combined knockdown/overexpression experiments (e.g., YAP knockdown with NUPR1 overexpression)

• Pharmacological pathway inhibition (e.g., AKT inhibitors) in combination with genetic NUPR1 manipulation

• Assessment of downstream targets through phosphorylation status analysis (p-YAP, p-AKT, p-p21)

These approaches collectively provide a comprehensive understanding of NUPR1's position within drug resistance mechanisms.

What methodological approaches are recommended for studying NUPR1's transcriptional regulatory functions?

Investigating NUPR1's role as a transcriptional regulator requires specialized methodologies. Chromatin Immunoprecipitation (ChIP) assays using validated NUPR1 antibodies are essential for identifying direct binding sites of NUPR1 on genomic DNA . For optimal ChIP results, researchers should use antibodies specifically validated for this application and implement appropriate sonication conditions to generate 200-500bp DNA fragments.

Transcriptome analysis through RNA-seq comparing control and NUPR1-knockdown or overexpressing cells provides comprehensive insights into the genes regulated by NUPR1. Bioinformatic analysis should focus on pathways related to cell migration, EMT, apoptosis, and drug resistance, as these have been implicated in NUPR1's biological functions .

For analysis of NUPR1's protein-protein interactions in transcriptional complexes, co-immunoprecipitation (Co-IP) using NUPR1 antibodies followed by mass spectrometry can identify binding partners that may mediate its transcriptional effects . When investigating NUPR1's interaction with YAP, researchers should consider the dynamic changes in these interactions following drug treatment or cellular stress conditions .

Reporter assays using promoter regions of NUPR1-regulated genes can provide functional validation of direct transcriptional regulation. For instance, constructs containing promoters of EMT-related genes (E-cadherin, N-cadherin, Vimentin) or apoptosis regulators (Bcl-2) can be used to assess NUPR1's direct regulatory impact .

How can NUPR1 antibodies be used to develop potential therapeutic strategies?

Exploiting NUPR1 antibodies for therapeutic development requires several strategic approaches. For target validation, immunohistochemistry with NUPR1 antibodies can be used to establish expression profiles across large patient cohorts, correlating expression with clinical outcomes to identify patient populations most likely to benefit from NUPR1-targeted therapies .

Researchers developing NUPR1 inhibition strategies should use NUPR1 antibodies to monitor target engagement and pathway modulation. This includes assessing changes in NUPR1 protein levels, subcellular localization, and downstream signaling effects after treatment with candidate compounds . When exploring combination therapy approaches, NUPR1 antibodies can help monitor changes in NUPR1 expression following treatment with conventional chemotherapeutics, identifying optimal timing for introduction of NUPR1-targeted agents .

For understanding resistance mechanisms to NUPR1-targeted therapies, researchers should monitor changes in NUPR1 expression, localization, and post-translational modifications in resistant cell populations using appropriate antibodies . In xenograft models, NUPR1 antibodies can be used for immunohistochemical analysis of tumor tissues to correlate treatment response with target modulation in vivo.

The following table summarizes potential therapeutic strategies targeting NUPR1 and the role of antibodies in their development:

How should researchers address non-specific binding issues with NUPR1 antibodies?

Non-specific binding can significantly compromise experimental results when using NUPR1 antibodies. To mitigate this issue, researchers should implement several optimization strategies. For Western blotting applications, increasing blocking stringency by using 5% BSA instead of milk and adding 0.1-0.3% Tween-20 to wash buffers can reduce background. Titrating antibody concentrations is essential, as NUPR1 antibodies may require different optimal dilutions than standard protocols suggest .

For immunohistochemistry and immunofluorescence applications, pre-adsorption of the primary antibody with the immunizing peptide (if available) can help identify and eliminate non-specific signals. Including additional blocking steps with normal serum from the secondary antibody host species can further reduce background. When interpreting results, researchers should be cautious about signals that appear in unexpected subcellular locations, as NUPR1 is primarily a nuclear protein .

In flow cytometry applications, additional washing steps and including isotype controls matched to the specific NUPR1 antibody being used are crucial. When performing functional studies involving NUPR1 knockdown, researchers should verify the specificity of their antibody by demonstrating reduced signal intensity in knockdown samples compared to controls .

What are the common pitfalls when quantifying NUPR1 expression levels?

Accurate quantification of NUPR1 expression presents several challenges that researchers must address. For qPCR-based quantification, selection of appropriate reference genes is critical, as NUPR1 expression can be affected by cellular stress conditions that might also alter commonly used housekeeping genes . Using multiple reference genes and validating their stability under experimental conditions improves reliability.

For protein-level quantification, NUPR1's small size (8.9 kDa) can lead to transfer inefficiencies during Western blotting, resulting in underestimation of expression levels . Using optimized transfer conditions for small proteins (lower voltage, longer time, or specialized membranes) and validating transfer efficiency with pre-stained markers in the appropriate molecular weight range improves accuracy.

When quantifying NUPR1 from clinical samples, heterogeneity within tumor tissues can lead to sampling bias. Multiple sampling from different tumor regions and correlating results with pathological examination helps ensure representative quantification . For immunohistochemical scoring, implementing standardized scoring systems that account for both staining intensity and percentage of positive cells provides more reliable quantitative data .

Researchers should also be aware that NUPR1 expression dynamics can change rapidly in response to experimental conditions, particularly drug treatments. Time-course analyses rather than single timepoint measurements provide a more comprehensive understanding of expression changes .

How can researchers overcome challenges in detecting NUPR1 in multiple species?

Working with NUPR1 across different species introduces challenges due to sequence variations that may affect antibody recognition. When selecting antibodies for cross-species applications, researchers should prioritize products validated across multiple species or those targeting highly conserved regions of NUPR1 . Consulting sequence alignments between species helps identify conserved epitopes that might serve as optimal antibody targets.

For Western blotting applications, researchers should adjust expected molecular weight parameters as NUPR1 size may vary slightly between species. Running positive control samples from each species of interest provides crucial reference points for band identification. When quantitative comparisons between species are required, validation of equal antibody affinity across species through titration experiments is essential .

In immunohistochemistry applications, optimization of antigen retrieval conditions may differ between species due to variations in tissue fixation responses and protein-protein interactions. Species-specific protocol modifications, particularly in blocking steps and antibody concentrations, should be empirically determined .

For researchers developing new models or working with less common species, preliminary validation using multiple detection methods (Western blotting, IHC, IF) provides stronger evidence of specific NUPR1 detection. When sequence information is available, epitope mapping helps predict which antibodies might perform consistently across species barriers.