ING4 Antibody

Basic Research Questions

• What is ING4 and what cellular functions does it regulate in experimental models?

  ING4 (Inhibitor of growth family, member 4) is a tumor suppressor protein implicated in numerous cellular processes including cell cycle arrest, apoptosis, cell migration, and angiogenesis . With a calculated molecular weight of 29 kDa (observed at 29-35 kDa in Western blots), ING4 functions as a component of the HBO1 complex which has histone H4-specific acetyltransferase activity . It may inhibit tumor progression by modulating the transcriptional output of signaling pathways regulating cell proliferation, and can suppress tumor angiogenesis through transcriptional repression of RELA/NFKB3 target genes when complexed with RELA .

  In experimental models, ING4 has demonstrated significant regulatory roles in:

  • NF-κB signaling pathway modulation

  • Inflammatory response suppression

  • Histone acetylation regulation

  • Angiogenesis inhibition

• What are the optimal applications for ING4 antibodies in molecular biology research?

  ING4 antibodies have been validated for multiple experimental applications with specific recommended dilutions:

  Application	Recommended Dilution	Validation Status
  Western Blot (WB)	1:500-1:4000 or 1 μg/mL	Validated in HEK-293, HeLa, C6 cells and rat brain tissue
  Immunohistochemistry (IHC)	1:50-1:500	Validated in mouse brain tissue and human samples
  Immunofluorescence (IF/ICC)	1:10-1:100	Validated
  Immunoprecipitation (IP)	1:200-1:1000	Validated
  ELISA	Variable by manufacturer	Validated

  For optimal results, antigen retrieval with TE buffer pH 9.0 is suggested for IHC applications, though citrate buffer pH 6.0 can be used as an alternative . Each antibody should be titrated in your specific experimental system to obtain optimal results.

• How should ING4 antibodies be stored and handled to maintain reactivity?

  To maintain optimal antibody performance, follow these storage guidelines:

  • Store at -20°C for long-term preservation

  • Aliquot to avoid repeated freeze/thaw cycles which can degrade antibody quality

  • Most commercial preparations are supplied in PBS buffer with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Typical shelf life/validity is 12 months when properly stored

  • Some smaller volume preparations (20μL) may contain 0.1% BSA as a stabilizer

  When shipping between laboratories, maintain cold chain integrity by using dry ice to prevent activity loss .

Intermediate Research Questions

• How can ING4 expression be effectively assessed in tissue microarrays and what scoring systems are recommended?

  For robust assessment of ING4 expression in tissue microarrays (TMAs), researchers should implement the following methodological approach:

  1. Antibody selection: Use polyclonal rabbit anti-ING4 antibody at 1:2000 dilution for optimal sensitivity

  2. Staining protocol:

     • Perform antigen retrieval (TE buffer pH 9.0 recommended)

     • Include negative controls (slides without primary antibody incubation)

     • Incubate with primary antibody at 4°C overnight

  3. Recommended scoring system:

     • Apply a semiquantitative immunoreactivity score (IRS) which is the product of staining intensity and percentage of immunopositive cells

     • Score intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

     • Score percentage: 1 (0-25%), 2 (26-50%), 3 (51-75%), 4 (76-100%)

     • Calculate IRS as the product of both scores (range: 0-12)

  4. Cutoff determination:

     • Use receiver-operator characteristic (ROC) analysis to obtain the optimum cutoff value

     • A cutoff value of 3 has shown the best predictive value for survival in colorectal cancer studies

     • Samples with IRS 0-3 can be classified as low expression and IRS 4-12 as high expression

  This standardized approach allows for reproducible assessment of ING4 expression across different laboratories and studies.

• What experimental models exist for studying ING4 function in inflammatory responses?

  Several validated experimental models are available for investigating ING4's role in inflammatory responses:

  1. Cellular models:

     • RAW 264.7 macrophage cell line with ING4 overexpression or knockdown via transient transfection with pcDNA3.1-ING4 or pLVX-shING4 plasmids

     • Primary macrophages isolated from Ing4-null mice

     • LPS-stimulated macrophages (typically using 100 ng/mL LPS for stimulation)

  2. In vivo models:

     • Ing4-deficient mouse model generated through gene targeting

     • LPS-induced sepsis mouse model (intraperitoneal LPS injection at 5 mg/kg or 15 mg/kg)

     • In vivo transfection of ING4 via caudal vein injection using Micropoly-transfecter Tissue Reagent

  3. Assessment methods:

     • Measurement of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) by ELISA

     • Evaluation of NF-κB pathway activation by assessing nuclear RelA levels

     • Analysis of histone H4 acetylation at specific promoters

     • Tissue damage assessment by hematoxylin-eosin staining

  These models have revealed that ING4 suppresses the production of cytokines in LPS-stimulated mice and that ING4-overexpressing mice are hyposensitive to LPS challenge with reduced organ injury .

• How do you validate the specificity of an ING4 antibody for experimental applications?

  A comprehensive validation approach for ING4 antibodies should include:

  1. Positive and negative control samples:

     • Use known positive samples: HEK-293 cells, HeLa cells, C6 cells, and rat brain tissue have been confirmed to express ING4

     • Include ING4 knockout/knockdown cells as negative controls

  2. Western blot validation:

     • Confirm single band at the expected molecular weight (29-35 kDa)

     • Test multiple cell lines to ensure consistent detection

     • Include competition assays with the immunizing peptide

  3. Immunoprecipitation confirmation:

     • Perform IP followed by Western blot to confirm antibody specificity

     • Verify with co-immunoprecipitation studies to detect known interaction partners like SIRT1

  4. Cross-reactivity testing:

     • Test reactivity across species (human, mouse, rat) if working with animal models

     • Evaluate potential cross-reactivity with other ING family members

  5. Immunohistochemical validation:

     • Compare staining patterns with published literature (primarily nuclear localization for ING4)

     • Include tissue microarrays with known differential expression patterns

     • Perform peptide blocking to confirm specificity

  For high-confidence results, validation across multiple applications and experimental systems is recommended.

Advanced Research Questions

• What mechanisms underlie ING4's regulation of NF-κB signaling and how can these be experimentally investigated?

  ING4 regulates NF-κB signaling through multiple mechanisms that can be experimentally investigated using the following approaches:

  1. IκBα promoter activation mechanism:

     • ING4 is required for robust activation of the IκBα promoter

     • LPS-stimulated Ing4-null cells show reduced levels of IκBα promoter H4 acetylation and IκB gene expression

     • Experimental approach: Chromatin immunoprecipitation (ChIP) assays to assess histone H4 acetylation at the IκBα promoter in wildtype vs. Ing4-null cells

  2. SIRT1 interaction pathway:

     • ING4 directly interacts with SIRT1 protein as confirmed by co-immunoprecipitation studies

     • Through this interaction, ING4 inhibits NF-κB signaling activation

     • Experimental approach: Co-immunoprecipitation with anti-SIRT1 and anti-ING4 antibodies, followed by functional assays with SIRT1 inhibitors/activators

  3. NF-κB nuclear translocation regulation:

     • ING4 blocks nuclear factor-kappa B (NF-κB) P65 nuclear translocation

     • ING4 restricts P65 acetylation at lysine 310 induced by LPS treatment

     • Experimental approach: Nuclear/cytoplasmic fractionation followed by Western blot for p65; immunofluorescence to track p65 localization

  4. RelA binding regulation:

     • Macrophages from Ing4-null mice have increased levels of nuclear p65/RelA protein

     • This results in increased RelA binding to NF-κB target promoters

     • Experimental approach: Electrophoretic mobility shift assay (EMSA) to assess DNA-protein binding affinity with specific NF-κB binding elements

  These mechanisms demonstrate that ING4 acts as a negative regulator of inflammation through multiple points of intervention in the NF-κB pathway, which can be systematically investigated using the approaches outlined above.

• How does ING4 regulate tumor angiogenesis and what experimental approaches can characterize this function?

  ING4 regulates tumor angiogenesis through multiple molecular mechanisms that can be experimentally characterized using these approaches:

  1. Sp1 transcriptional regulation pathway:

     • ING4 inhibits angiogenesis by suppressing Sp1 expression and transcriptional activity through ubiquitin degradation

     • This leads to downregulation of Sp1 downstream pro-angiogenic genes MMP-2 and COX-2

     • Experimental approach: EMSA to determine Sp1-binding affinity to Sp1-responsive DNA elements; quantify MMP-2 and COX-2 expression by qRT-PCR and Western blot

  2. p21-dependent Sp1 degradation mechanism:

     • ING4 can induce p21 expression to inhibit phosphorylation activity of cyclin/CDK2 complexes

     • This triggers Sp1 degradation despite p53 status

     • Experimental approach: Combined ING4 knockdown and p21 overexpression to demonstrate rescue effects on Sp1 expression

  3. In vivo angiogenesis models:

     • Characterize vessel formation in ING4-overexpressing vs. control tumors

     • Experimental approach: Quantify microvessel density using CD31/PECAM-1 immunostaining; perform in vivo Matrigel plug assays

  4. HIF regulation pathway:

     • ING4 represses hypoxia-inducible factor (HIF) activity by interacting with HIF prolyl hydroxylase 2 (EGLN1)

     • Experimental approach: Co-immunoprecipitation of ING4 with HIF-1α and EGLN1; measure HIF target genes under hypoxic conditions

  5. Clinical correlation approaches:

     • Analyze ING4 expression in patient samples in relation to angiogenesis markers

     • Experimental approach: Immunohistochemical analysis of tissue microarrays for ING4 and angiogenesis markers (CD31, VEGF, MMP-2, COX-2)

  These studies have demonstrated that reduced ING4 expression in colorectal cancer results in increased angiogenesis, contributing to metastasis and poor prognosis .

• What are the mechanistic differences between ING4's regulation of inflammation in p53-wild type versus p53-deficient systems?

  ING4 exhibits both p53-dependent and p53-independent regulatory mechanisms in inflammation control, which can be experimentally distinguished:

  1. p21 regulation pathway:

     • ING4 positively regulates p21 expression at both mRNA and protein levels through the induction of p21 promoter activation

     • Significantly, this regulation occurs regardless of p53 status in both p53-wild and p53-deficient CRC cells

     • This finding indicates that ING4 can exert anti-inflammatory effects through p21 induction even in p53-mutated or deficient tumors, which are common in cancer

  2. Cyclin-CDK2 complex regulation:

     • ING4 reduces the expressions of Sp1 and cyclin-CDK2 complex phosphorylation activity in spite of p53 expression

     • The inhibition of cyclin/CDK2 phosphorylation activity by ING4-induced p21 triggers Sp1 degradation in both p53-positive and p53-negative contexts

     • This regulatory axis represents a p53-independent mechanism for controlling inflammation-related transcription factors

  3. Experimental verification approaches:

     • Comparative studies using p53-wild type and p53-null cell lines (like HCT116 p53+/+ and HCT116 p53-/- colorectal cancer cells)

     • Analysis of ING4's effects on histone acetylation patterns at inflammatory gene promoters in both systems

     • Assessment of NF-κB pathway activation markers in response to ING4 manipulation in p53-positive versus p53-negative backgrounds

  4. Chromatin remodeling activities:

     • ING4 may employ different histone acetyltransferase or deacetylase partners depending on p53 status

     • In both contexts, ING4 facilitates proper histone H4 acetylation at specific promoters, but the underlying molecular machinery may differ

  These findings have significant implications for targeting ING4-related pathways in cancer therapies, particularly in tumors with p53 mutations, suggesting that ING4-based interventions may still be effective regardless of p53 status .

• How can ChIP-seq and other genomic approaches be optimized to investigate ING4's role in histone modification and chromatin regulation?

  Optimizing genomic approaches to study ING4's chromatin regulatory functions requires specialized methodologies:

  1. ChIP-seq protocol optimization for ING4:

     • Crosslinking optimization: Use dual crosslinking with DSG (disuccinimidyl glutarate) followed by formaldehyde for improved capture of protein-DNA interactions

     • Sonication parameters: Optimize to yield 200-300bp fragments for high-resolution mapping

     • Antibody selection: Use ChIP-grade ING4 antibodies validated for immunoprecipitation

     • Controls: Include IgG control and ING4-knockout cells as negative controls

  2. Sequential ChIP (ChIP-reChIP) approach:

     • Implement to detect co-occupancy of ING4 with known interacting partners:

       • ING4/HBO1 (histone acetyltransferase complex)

       • ING4/SIRT1 (identified interaction partner)

       • ING4/RelA (for inflammatory response genes)

  3. Integrated genomic approaches:

     • Combine ChIP-seq with RNA-seq to correlate ING4 binding with transcriptional outcomes

     • Perform ATAC-seq to identify chromatin accessibility changes related to ING4 activity

     • Use CUT&RUN or CUT&Tag for higher sensitivity detection of ING4 binding sites

  4. Histone modification analysis:

     • Focus on H4K16ac and H3K14ac marks associated with HBO1 complex activity

     • Analyze H3K9ac and H3K27ac as markers of active enhancers and promoters

     • Perform sequential ChIP for ING4 followed by specific histone mark antibodies

  5. Data analysis considerations:

     • Use motif discovery to identify DNA sequences preferentially bound by ING4-containing complexes

     • Perform pathway enrichment analysis focusing on inflammatory and angiogenesis pathways

     • Compare binding patterns between normal and inflammatory conditions (e.g., with/without LPS stimulation)

  These approaches have revealed that ING4 is required for proper histone H4 acetylation at select promoters, including the IκBα promoter , and that its chromatin regulatory activities are central to its roles in inflammation and tumor suppression.