IFRD1 Antibody

What is IFRD1 and why is it important in research?

IFRD1 (interferon-related developmental regulator 1) is a transcriptional coactivator/repressor that controls gene expression by interacting with transcription factors or histone deacetylase (HDAC) complexes . It plays critical roles in multiple cellular processes including osteoclast differentiation, stress response regulation, and epithelial homeostasis . IFRD1 expression is upregulated in response to acute tissue injuries, various growth factors, and cytokines . Its importance in research stems from its implication in several pathological conditions, including cystic fibrosis through the regulation of neutrophil effector cells, and as a candidate gene for autosomal-dominant sensory/motor neuropathy with ataxia . IFRD1 is also involved in bone remodeling via an Ifrd1/NF-κB/NFATc1 axis, making it a potential therapeutic target for bone diseases .

What are the key characteristics of IFRD1 antibodies?

IFRD1 antibodies are immunological reagents designed to specifically bind to IFRD1 protein epitopes. The commercially available antibody (12939-1-AP) is a rabbit polyclonal IgG that targets IFRD1 in Western blot (WB), immunohistochemistry (IHC), and ELISA applications . This antibody demonstrates reactivity with human samples and is generated using an IFRD1 fusion protein (Ag3988) as the immunogen . The antibody recognizes IFRD1 with a calculated molecular weight of 50 kDa (451 amino acids) and an observed molecular weight of 51 kDa in experimental settings . It is supplied in liquid form, purified through antigen affinity purification, and stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

How should IFRD1 antibodies be stored and handled for optimal results?

For optimal preservation of antibody activity, IFRD1 antibodies should be stored at -20°C, where they remain stable for one year after shipment . Importantly, aliquoting is unnecessary for -20°C storage, which simplifies laboratory handling protocols . The 20μl size preparations contain 0.1% BSA as a stabilizer . When working with the antibody, avoid repeated freeze-thaw cycles as these can degrade antibody quality and compromise experimental results. During experiments, maintain the antibody on ice when in use and return it to -20°C promptly after completing procedures. When pipetting, use sterile technique to prevent contamination that could affect antibody performance and experimental outcomes.

What are the recommended dilutions for different applications of IFRD1 antibody?

The optimal working dilutions for IFRD1 antibody vary depending on the specific application:

It is essential to note that these dilutions are guidelines, and researchers should titrate the antibody in each testing system to obtain optimal results as the appropriate dilution may be sample-dependent . For Western blotting applications, researchers typically start with a 1:1000 dilution and adjust based on signal intensity. For IHC applications, antigen retrieval is suggested with TE buffer pH 9.0, though citrate buffer pH 6.0 may alternatively be used . Standardizing protocols within your laboratory for specific sample types will improve reproducibility across experiments.

How can I validate IFRD1 antibody specificity for my experiments?

Validating antibody specificity is critical for ensuring reliable experimental results. For IFRD1 antibody validation, employ multiple complementary approaches:

• Positive controls: Use samples known to express IFRD1, such as HeLa cells or human skeletal muscle tissue, which have been confirmed to show positive Western blot results with this antibody .

• Immunohistochemistry validation: Human intrahepatic cholangiocarcinoma tissue has been verified as a positive control for IHC applications .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of approximately 51 kDa .

• siRNA knockdown: Perform siRNA-mediated knockdown of IFRD1 in your experimental system and verify reduced antibody signal. This approach was successfully used in studies examining RANKL-induced IFRD1 expression .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide to demonstrate signal reduction in Western blots.

• Secondary antibody controls: Run controls omitting the primary antibody to identify potential non-specific binding of the secondary antibody.

What cell lines and tissue samples are suitable for IFRD1 expression studies?

Several cell lines and tissue samples have been validated for IFRD1 expression studies:

When selecting experimental systems, consider that IFRD1 expression can be significantly modulated by stress conditions or specific stimuli. For instance, tunicamycin treatment increases IFRD1 mRNA and protein levels in human kidney epithelial cells through stabilization of the mRNA . Similarly, RANKL treatment induces IFRD1 expression in preosteoclasts through the activator protein 1 (AP-1) pathway . These inducible expression systems can provide valuable experimental models for studying IFRD1 regulation and function.

What are common issues with Western blot detection of IFRD1 and how can they be resolved?

Several challenges may arise when detecting IFRD1 via Western blot:

• Weak or absent signal: This could be due to insufficient protein loading, antibody dilution that is too high, or degraded samples. Solutions include:

  • Increase protein loading (20-40 μg total protein)

  • Optimize antibody concentration (start with 1:500 dilution)

  • Ensure proper sample preparation with protease inhibitors (APMSF, leupeptin, pepstatin A, antipain at 1 μg/ml)

  • Extended exposure times for chemiluminescent detection

• Multiple bands or non-specific binding: This may indicate cross-reactivity or protein degradation. Address by:

  • Including proper negative controls

  • Optimizing blocking conditions (5% non-fat milk or BSA)

  • Using freshly prepared samples with complete protease inhibitor cocktail

  • Increasing washing stringency between antibody incubations

• High background: Can result from insufficient blocking or washing. Improve by:

  • Extending blocking time (1-2 hours at room temperature)

  • Using additional wash steps with increased TBST volume

  • Decreasing secondary antibody concentration

• Inconsistent results: May stem from variable IFRD1 expression under different cellular conditions. Standardize by:

  • Maintaining consistent cell culture conditions

  • Using positive control samples (HeLa cells)

  • Normalizing results to housekeeping proteins (GAPDH or β-actin)

Following established protocols specific for IFRD1 antibody (12939-1-AP) and proper controls will significantly improve Western blot results .

How can I optimize immunohistochemical detection of IFRD1 in tissue sections?

Optimizing IHC detection of IFRD1 requires careful attention to several experimental parameters:

• Antigen retrieval: The suggested protocol recommends TE buffer at pH 9.0, though citrate buffer at pH 6.0 may also be effective as an alternative . Comparing both methods for your specific tissue is advisable.

• Antibody dilution: Start with a mid-range dilution (1:200) within the recommended range (1:50-1:500) , then adjust based on signal-to-noise ratio. Titration experiments across multiple dilutions are recommended for each new tissue type.

• Incubation conditions:

  • Primary antibody: Incubate overnight at 4°C to maximize specific binding

  • Secondary antibody: 1-2 hours at room temperature

  • Allow sections to reach room temperature before starting the protocol

• Controls:

  • Positive control: Human intrahepatic cholangiocarcinoma tissue

  • Negative controls: Omit primary antibody or use non-immune IgG

  • Competitive peptide blocking to demonstrate specificity

• Signal development:

  • Optimize DAB (3,3′-diaminobenzidine) exposure time

  • Consider signal amplification systems for low-abundance expression

  • Use hematoxylin counterstaining to provide cellular context

• Troubleshooting specific issues:

  • High background: Increase blocking time or concentration

  • Weak signal: Extend antibody incubation time or increase concentration

  • Non-specific staining: Additional washing steps or more dilute antibody

Document all optimization steps methodically to establish a reproducible protocol for your specific tissue and research questions.

What considerations are important when using IFRD1 antibody for co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments with IFRD1 antibody, several critical factors should be considered:

• Antibody compatibility: The IFRD1 antibody (12939-1-AP) has been successfully used for co-IP studies in human bladder cancer cell line 5637 , demonstrating its utility for protein interaction studies.

• Lysis buffer composition: Use a buffer that preserves protein-protein interactions while effectively solubilizing membrane-associated proteins. A typical buffer might include:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • 1 mM EDTA

  • Protease inhibitor cocktail

  • Phosphatase inhibitors (if studying phosphorylation-dependent interactions)

• Blocking non-specific interactions: Pre-clear lysates with Protein A/G beads and appropriate control IgG to reduce background.

• Controls:

  • IgG control: Essential for distinguishing specific from non-specific interactions

  • Input control: Include 5-10% of pre-immunoprecipitation lysate

  • Reverse co-IP: Confirm interactions by immunoprecipitating suspected binding partners

• Detection methods:

  • Western blotting: For targeted validation of specific interactions

  • Mass spectrometry: For unbiased discovery of interaction partners

• Data interpretation: When analyzing MS/MS results, focus on proteins exclusively detected in IFRD1 precipitates but not in IgG controls. In one study, 943 proteins were found to exclusively interact with IFRD1 .

• Validation of novel interactions: Confirm key interactions using complementary methods such as proximity ligation assay, FRET, or reciprocal co-IP.

The successful identification of IFRD1-interacting proteins provides valuable insights into its functional roles in various cellular processes.

How does IFRD1 expression change under different cellular stress conditions?

IFRD1 expression exhibits dynamic regulation under various cellular stress conditions, serving as an important stress-response mediator:

• ER stress response: Tunicamycin treatment, which induces endoplasmic reticulum stress through the unfolded protein response (UPR), significantly increases IFRD1 mRNA and protein levels in human kidney epithelial cells . This increase is detectable within 4 hours of treatment and persists through at least 8 hours .

• Post-transcriptional regulation: The increase in IFRD1 mRNA during stress is not due to enhanced transcription but rather to mRNA stabilization. In unstressed cells, IFRD1 mRNA has a half-life of approximately 2 hours, which extends to almost 6 hours following tunicamycin treatment .

• Translational control mechanism: IFRD1 mRNA stability is regulated through an upstream open reading frame (uORF) that represses translation of the major ORF in resting cells. During stress response, phosphorylation of eIF2α inhibits translational initiation, leading to mRNA stabilization .

• Transcript-specific regulation: Two separate transcripts (TR1 and TR2) have been reported for the IFRD1 gene, differing in sequence within the 5'-leader region. Interestingly, only TR1 shows stress-dependent upregulation, while TR2 remains undetectable or unresponsive to stress stimuli .

• Acute tissue injury response: Beyond chemical stressors, IFRD1 expression is upregulated in vivo by acute tissue injuries, including cerebral ischemia/reperfusion and muscle trauma .

These findings highlight IFRD1's role as a stress-sensitive regulator whose expression is controlled through sophisticated post-transcriptional mechanisms, providing a rapid response to cellular stress conditions.

What is the role of IFRD1 in regulating osteoclast differentiation and bone remodeling?

IFRD1 plays a crucial role in osteoclast differentiation and bone remodeling through several molecular mechanisms:

Understanding IFRD1's role in osteoclast differentiation provides important insights into the molecular mechanisms controlling bone homeostasis and potential therapeutic approaches for bone disorders.

How does IFRD1 interact with other cellular proteins to exert its biological functions?

IFRD1 functions as a transcriptional modulator through extensive protein-protein interactions with various cellular components:

• Interactome complexity: Proteomic analysis using co-immunoprecipitation followed by tandem mass spectrometry (MS/MS) in human bladder cancer cell line 5637 identified 943 proteins that exclusively interacted with IFRD1 . This extensive interaction network highlights IFRD1's involvement in multiple cellular processes.

• HDAC interactions: IFRD1 interacts with histone deacetylase (HDAC) complexes to regulate gene expression . This interaction is functionally important, as IFRD1 deficiency inhibits HDAC-dependent deacetylation of the NF-κB subunit p65 at residues K122 and K123 .

• Transcription factor interactions: IFRD1 can function as both a transcriptional coactivator and corepressor by interacting with various transcription factors . Its interaction with NF-κB is particularly important for regulating the expression of NFATc1, a critical regulator of osteoclastogenesis .

• Cell type-specific interactions: The functional outcomes of IFRD1 interactions may vary depending on the cellular context:

  • In osteoclast precursors, IFRD1 promotes RANKL-induced differentiation through NF-κB/NFATc1 pathway modulation

  • In bladder epithelial cells, IFRD1 is required for maintaining epithelial homeostasis through interactions with components of the bladder interactome

  • In kidney epithelial cells, IFRD1 participates in stress response pathways

• Regulatory mechanism: IFRD1 can modulate protein function through influencing post-translational modifications, as demonstrated by its effect on p65 acetylation status .

• Temporal dynamics: The composition of the IFRD1 interactome may change in response to cellular conditions, such as stress or differentiation signals, allowing context-specific regulation of cellular processes.

Understanding this complex interactome provides insights into how a single protein can participate in diverse cellular processes and contribute to tissue-specific functions across different physiological contexts.

What experimental approaches can detect post-transcriptional regulation of IFRD1 expression?

Investigating the post-transcriptional regulation of IFRD1 requires sophisticated experimental approaches targeting various aspects of RNA processing and translation:

• mRNA half-life determination:

  • Actinomycin D chase experiments have revealed that IFRD1 mRNA half-life increases from approximately 2 hours in resting cells to almost 6 hours following tunicamycin treatment

  • Methodology: Treat cells with actinomycin D to block transcription, then measure residual IFRD1 mRNA levels at various time points using real-time RT-PCR

• Transcript-specific analysis:

  • Real-time RT-PCR with primers specific for each transcript variant (TR1 and TR2) can distinguish their differential regulation

  • Results show that TR1 mRNA levels increase in tunicamycin-treated cells, while TR2 mRNA remains undetectable

• Primary transcript quantification:

  • Primers spanning intron-exon junctions can measure the abundance of primary transcripts as an indicator of transcription rate

  • This approach demonstrated that tunicamycin treatment did not increase IFRD1 primary transcript levels despite elevated mature mRNA, confirming post-transcriptional regulation

• Transcription-independent expression systems:

  • Placing IFRD1 sequences under control of a tetracycline-regulated promoter (tet-off) allows examination of post-transcriptional effects independent of the endogenous gene promoter

  • This system revealed that TR1 mRNA and protein increased with tunicamycin treatment while TR2 showed no sensitivity

• Upstream ORF (uORF) analysis:

  • The instability mechanism of IFRD1 mRNA involves translation of an upstream open reading frame that represses translation of the major ORF

  • Investigating the role of uORF requires mutational analysis of the uORF sequence, length, and translation initiation contexts

• Translation mechanism studies:

  • IFRD1 translation involves both leaky scanning at the upstream AUG codon and re-initiation at the major AUG codon

  • The stress-induced stabilization mechanism depends upon UPF1 and is linked to phosphorylation of eIF2α

These sophisticated approaches collectively provide a comprehensive understanding of the complex post-transcriptional mechanisms regulating IFRD1 expression during cellular stress responses.

What are emerging applications for IFRD1 antibodies in disease research?

IFRD1 antibodies are becoming increasingly valuable tools in investigating various disease contexts:

• Bone disorders: Given IFRD1's critical role in osteoclast differentiation through the Ifrd1/NF-κB/NFATc1 axis, IFRD1 antibodies can help evaluate its expression and molecular interactions in pathological bone loss conditions, including osteoporosis and rheumatoid arthritis . The prevention of RANKL-induced bone loss in Ifrd1-deficient mice suggests potential therapeutic applications targeting this pathway .

• Bladder epithelial dysfunction: Recent findings demonstrating IFRD1's requirement for bladder epithelial homeostasis highlight its potential involvement in bladder pathologies . IFRD1 antibodies could be instrumental in investigating changes in expression or localization in conditions affecting urothelial function.

• Stress-related cellular pathologies: As a stress-sensitive regulator whose expression changes during the unfolded protein response, IFRD1 may participate in conditions involving cellular stress, such as neurodegenerative diseases, ischemia/reperfusion injury, and certain metabolic disorders . Antibody-based detection could track IFRD1 dynamics during disease progression.

• Cystic fibrosis: IFRD1 has been implicated in the pathophysiology of cystic fibrosis through regulation of neutrophil effector cell function . Antibodies targeting IFRD1 could help elucidate its role in inflammatory processes associated with this condition.

• Neurological disorders: As a candidate gene for autosomal-dominant sensory/motor neuropathy with ataxia, IFRD1 antibodies may contribute to understanding its involvement in neurological function and dysfunction .

• Cancer research: The detection of IFRD1 in bladder cancer cell lines and its extensive protein interaction network suggest potential roles in cancer biology . Immunohistochemical analysis using IFRD1 antibodies could assess its expression patterns across tumor types and stages.

These emerging applications highlight the potential of IFRD1 antibodies to advance our understanding of disease mechanisms and identify novel therapeutic targets across multiple pathological conditions.

What methodological advances could improve IFRD1 research?

Several methodological advances could significantly enhance IFRD1 research:

• Development of monoclonal antibodies: While polyclonal antibodies like 12939-1-AP provide valuable research tools, developing highly specific monoclonal antibodies against distinct epitopes of IFRD1 would enable more precise detection of different protein isoforms and post-translational modifications.

• Phospho-specific antibodies: Given the importance of protein phosphorylation in cellular signaling, antibodies that specifically recognize phosphorylated forms of IFRD1 could provide insights into its activation status under different conditions.

• CRISPR/Cas9-mediated genome editing:

  • Generation of IFRD1 knockout cell lines across different tissue types

  • Creation of cells expressing tagged endogenous IFRD1 for improved detection and purification

  • Introduction of specific mutations to evaluate the functional significance of particular domains or residues

• Live-cell imaging techniques:

  • Development of fluorescently tagged IFRD1 constructs that maintain native function

  • Application of advanced microscopy techniques (FRAP, FRET, super-resolution) to study IFRD1 dynamics and interactions in real-time

  • Optogenetic approaches to precisely control IFRD1 activity in specific cellular compartments

• Integrative multi-omics approaches:

  • Combining proteomics, transcriptomics, and ChIP-seq to comprehensively map IFRD1's regulatory networks

  • Analysis of IFRD1-associated ribonucleoprotein complexes to better understand its role in post-transcriptional regulation

  • Global mapping of histone modifications influenced by IFRD1-HDAC interactions

• Structural biology approaches:

  • Determination of IFRD1 crystal structure alone and in complex with key interacting partners

  • Structure-guided design of small molecule modulators of IFRD1 function

  • Analysis of conformational changes associated with IFRD1 activation

• High-throughput screening platforms:

  • Development of reporter assays to monitor IFRD1 activity

  • Screening for compounds that modulate IFRD1 expression or function

  • Identification of synthetic lethal interactions in IFRD1-deficient backgrounds

These methodological advances would significantly expand our understanding of IFRD1 biology and potentially lead to new therapeutic strategies targeting IFRD1-dependent pathways in various diseases.