IFRD1 Antibody,HRP conjugated

What is IFRD1 and what are its key biological functions?

IFRD1 (Interferon-related developmental regulator 1), also known as Nerve growth factor-inducible protein PC4, regulates gene activity in proliferative and differentiative pathways induced by NGF. It functions as an autocrine factor that may attenuate or amplify initial ligand-induced signals . Research has revealed that IFRD1 plays critical roles in immune modulation by suppressing cytokine/chemokine production in HPV-positive cells and promoting tumor cell survival under metabolic stress conditions . Methodologically, researchers can study these functions using techniques like siRNA knockdown followed by cytokine expression analysis, as demonstrated in HPV-infected keratinocyte models .

How do HRP-conjugated IFRD1 antibodies differ from unconjugated versions?

While standard IFRD1 antibodies require secondary detection reagents, HRP-conjugated versions offer direct detection capabilities through enzymatic reactions with substrates. For methodology implementation, this eliminates the need for secondary antibody incubation steps, reducing experimental time and potential cross-reactivity issues. When working with HRP-conjugated IFRD1 antibodies, researchers should carefully optimize substrate choice (TMB, DAB, or chemiluminescent reagents) based on the required sensitivity and detection method. The conjugation process may affect antibody binding characteristics compared to unconjugated versions, potentially necessitating different dilution factors than specified for the parent antibody.

Which experimental applications are validated for IFRD1 antibodies?

Based on available research, IFRD1 antibodies have been successfully applied in Western blotting (WB), immunohistochemistry on paraffin-embedded samples (IHC-P), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence (IF) . For rigorous experimental design, researchers should consider that most validated antibodies are rabbit polyclonal antibodies with confirmed reactivity to human and mouse IFRD1 . When conducting IHC experiments, nuclear localization of IFRD1 has been observed specifically in HPV-infected cells positive for E2, but not in transformed keratinocytes (identified through p16 staining) or healthy tissue .

What expression patterns should researchers expect when studying IFRD1?

IFRD1 expression is dynamically regulated under various conditions, requiring careful experimental controls. In HPV-related research, IFRD1 gene expression and protein levels are significantly elevated in HPV16-positive keratinocyte cultures compared to uninfected controls . Methodologically, this can be verified through reverse transcriptase-quantitative PCR (RT-qPCR) and western blotting . Expression changes are observable within 2 days of HPV16 infection in undifferentiated keratinocytes . Additionally, researchers should account for IFRD1 induction during glutamine starvation in hepatocellular carcinoma models, where it plays a role in adaptive survival mechanisms .

How should researchers optimize Western blotting protocols for IFRD1 detection?

For optimal Western blot detection of IFRD1, researchers should implement a methodological approach that accounts for potential variability in expression levels. Based on published research, effective protocols include standard SDS-PAGE separation followed by transfer to nitrocellulose or PVDF membranes . When using HRP-conjugated antibodies, it's critical to optimize blocking conditions (typically 5% non-fat milk or BSA) to minimize background. For analyzing how IFRD1 levels change in response to treatments like EGFR inhibition, researchers should perform relative density analysis comparing IFRD1 levels to appropriate housekeeping controls . The detection methodology should also account for potential changes in associated proteins like RelA and acetylated RelA K310 when studying IFRD1's functional impacts .

What controls are essential when using IFRD1 antibodies in research?

Rigorous experimental design requires multiple controls to ensure reliable interpretation of IFRD1 antibody data. Critical methodological controls include:

• Knockdown validation: siRNA against IFRD1 provides essential negative controls, as demonstrated in HPV16+ keratinocyte studies where IFRD1 knockdown resulted in increased levels of acetylated RelA K310 .

• Treatment controls: When studying pathway influences, appropriate controls are necessary. For example, when examining EGFR signaling effects on IFRD1, using control antibodies like rituximab (anti-CD20) alongside the test antibody cetuximab provides critical comparison data .

• Pathway inhibitor controls: For mechanistic studies, selective inhibitors of downstream pathways (mTOR, MEK1, RAF, PI3K, JNK) help delineate specific pathways regulating IFRD1 expression .

• Positive detection controls: Recombinant IFRD1 protein or lysates from cells known to express high IFRD1 levels (e.g., HPV16+ keratinocytes) should be included when establishing detection protocols.

How can researchers use IFRD1 antibodies to study its role in immune regulation?

IFRD1 plays a significant role in suppressing immune responses, particularly in HPV-infected cells. Methodologically, researchers can use IFRD1 antibodies to investigate:

• NFκB pathway interactions: IFRD1 forms complexes with HDAC1 and HDAC3, leading to deacetylation of RelA at lysine 310 . This can be studied using co-immunoprecipitation with IFRD1 antibodies followed by western blotting for associated proteins.

• Cytokine modulation: IFRD1 knockdown in HPV16+ keratinocytes results in higher basal expression and secretion of cytokines, especially when stimulated with IFN-γ and TNF-α . Researchers can use ELISA or multiplex cytokine assays to quantify these changes.

• Immune cell recruitment: The dampening effect of IFRD1 on NFκB-regulated cytokine expression affects the ability of cells to attract peripheral blood mononuclear cells (PBMCs) . Migration assays can be used alongside IFRD1 manipulation to assess functional outcomes.

The methodological approach should include pathway stimulation (e.g., with poly(I:C), IFN-γ, TNF-α) to fully reveal IFRD1's regulatory effects .

What signaling pathways regulate IFRD1 expression that researchers should consider?

IFRD1 expression is regulated through multiple signaling pathways, with EGFR signaling playing a particularly important role. For comprehensive mechanistic studies, researchers should consider:

Methodologically, studying these pathways requires selective inhibitors combined with IFRD1 expression analysis at both protein (Western blot) and mRNA (RT-qPCR) levels . Researchers should design dose-response experiments to determine the relative contribution of each pathway to IFRD1 regulation.

How can IFRD1 antibodies be used to investigate its role in cancer metabolism?

Recent research has revealed IFRD1's role in promoting tumor cell survival during glutamine deprivation, particularly in hepatocellular carcinoma . Methodological approaches to study this function include:

• Autophagy pathway analysis: IFRD1 inhibits autophagy by promoting the proteasomal degradation of the key autophagy regulator ATG14 in a TRIM21-dependent manner . Researchers can use IFRD1 antibodies in combination with autophagy markers to track this relationship.

• Chromatin regulation studies: IFRD1 depletion triggers nucleophilic degradation of histone H1.0, leading to enhanced chromatin accessibility and increased ribosome and protein biosynthesis . Chromatin immunoprecipitation (ChIP) using IFRD1 antibodies can help map its genomic interactions.

• Therapeutic targeting models: IFRD1 depletion synergizes with glutaminase-1 inhibitors like CB-839 in preclinical HCC models . Researchers can use IFRD1 antibodies to confirm knockdown efficiency and monitor expression changes during treatment.

What are common technical challenges when using HRP-conjugated antibodies for IFRD1 detection?

When working with HRP-conjugated IFRD1 antibodies, researchers may encounter several technical challenges requiring methodological solutions:

• Signal specificity: Validate specificity by comparing detection in IFRD1-positive tissues (e.g., HPV-infected cells) versus negative controls (E2 and p16 negative healthy tissue) .

• Subcellular localization variability: IFRD1 can display nuclear localization in HPV-infected cells positive for E2, but shows different patterns in transformed cells . Different fixation and permeabilization protocols may be needed depending on the cellular context.

• Antibody selection: Different epitopes within IFRD1 may be differentially accessible. Available antibodies target different regions, such as amino acids 1-250 of human IFRD1 , which may affect detection efficiency in different applications.

• Signal amplification needs: For low-abundance detection, HRP-conjugated antibodies may require additional signal amplification through tyramide signal amplification (TSA) or other enhanced chemiluminescence methods.

How can researchers verify IFRD1 antibody specificity in their experimental systems?

Methodological approaches to verify IFRD1 antibody specificity should include:

• Knockdown controls: siRNA or shRNA against IFRD1, as demonstrated in HPV16+ keratinocyte studies, provides essential negative controls .

• Recombinant protein controls: Using purified IFRD1 protein fragments for peptide competition assays can verify binding specificity.

• Multiple antibody comparison: Using antibodies targeting different IFRD1 epitopes and comparing detection patterns helps confirm target authenticity.

• Correlation with mRNA expression: Comparing protein detection with mRNA levels measured by RT-qPCR provides additional validation .

• Knockout models: Where available, IFRD1 knockout cell lines or tissues provide definitive negative controls.

What are recommended dilutions and processing parameters for different applications?

Based on available research and commercial antibody specifications, methodology recommendations include:

Note that optimal dilutions should be determined empirically for each specific antibody and experimental system. For HRP-conjugated antibodies, dilutions may differ from those recommended for unconjugated primary antibodies.

How might IFRD1 antibodies be used in therapeutic development research?

IFRD1's roles in immune suppression and cancer cell survival make it a potential therapeutic target. Methodological approaches for therapeutic development research include:

• Target validation: Using HRP-conjugated IFRD1 antibodies for immunohistochemistry to assess expression in patient samples can help identify potential responders to IFRD1-targeted therapies.

• Combination therapy models: Research demonstrates that IFRD1 depletion synergizes with glutaminase-1 inhibitors like CB-839 in HCC models . This suggests methodological approaches combining IFRD1 targeting with metabolic therapies.

• Immune restoration strategies: Given IFRD1's role in suppressing immune responses in HPV-infected cells , antibodies can help assess whether IFRD1 inhibition restores immune surveillance.

• Biomarker development: HRP-conjugated IFRD1 antibodies could be developed for diagnostic or prognostic use, particularly in HPV-associated cancers where IFRD1 upregulation occurs .

What emerging technologies might enhance IFRD1 research using antibodies?

Advanced methodological approaches that could enhance IFRD1 research include:

• Single-cell protein analysis: Techniques like mass cytometry (CyTOF) with metal-conjugated IFRD1 antibodies would allow high-dimensional analysis of IFRD1 expression in heterogeneous cell populations.

• Proximity labeling: BioID or APEX2 fusions with IFRD1 combined with antibody-based detection can map the IFRD1 interactome in different cellular contexts.

• Super-resolution microscopy: Combining IFRD1 antibodies with techniques like STORM or STED microscopy could reveal subcellular localization details beyond conventional microscopy.

• Spatial transcriptomics integration: Correlating IFRD1 protein expression (via antibody detection) with spatial transcriptomics would provide insights into its functional roles in tissue architecture.