NKIRAS2 Antibody

What are the validated applications for NKIRAS2 antibodies and their optimal working conditions?

NKIRAS2 antibodies have been validated for multiple applications with specific recommended dilutions as summarized in the following table:

It's critical to optimize antibody concentration for each specific experimental setup and sample type . When selecting antibodies, consider whether polyclonal or monoclonal antibodies are more appropriate for your specific research questions.

What is the optimal Western blot protocol for detecting NKIRAS2?

For optimal NKIRAS2 detection by Western blot, follow this methodological approach:

• Sample preparation:

  • Use RIPA buffer containing protease inhibitors

  • For cell lines (HEK-293, HepG2, U2OS), harvest at 70-80% confluence

  • Lyse cells on ice for 30 minutes with periodic vortexing

  • Centrifuge at 14,000g for 15 minutes at 4°C and collect supernatant

• SDS-PAGE and transfer:

  • Load 20-30 μg of total protein per lane on 12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membrane using standard methods

  • Verify transfer efficiency with Ponceau S staining

• Immunoblotting:

  • Block with 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with primary anti-NKIRAS2 antibody (1:500-1:1000) overnight at 4°C

  • Wash membrane 3-5 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000)

  • Wash thoroughly and develop using ECL substrate

• Controls and verification:

  • Positive controls: HEK-293 or HepG2 cell lysates (express detectable NKIRAS2)

  • Size verification: Expected band at 21-22 kDa

  • Specificity control: Pre-incubation with blocking peptide should eliminate specific signals

Note that while the calculated molecular weight of NKIRAS2 is 21.5 kDa, some researchers have observed bands at different sizes, possibly due to post-translational modifications or isoforms.

What evidence supports NKIRAS2's role in tumor suppression?

Multiple experimental approaches have demonstrated NKIRAS2's tumor suppressive function:

• In osteosarcoma (OS):

  • NKIRAS2 mRNA expression is reduced in OS cells while remaining highly expressed in normal osteoblast cell lines

  • NKIRAS2 knockdown promoted OS cell proliferation while overexpression inhibited proliferation

  • Transwell assays showed that NKIRAS2 significantly inhibited the invasive capacity of OS cells

  • The overexpression of NKIRAS2 inhibited OS progression both in vivo and in vitro

• In skin cancer model:

  • K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by DMBA/TPA treatment

  • This suggests NKIRAS2 functions as a tumor suppressor in follicle bulges

These findings provide compelling evidence that NKIRAS2 can inhibit tumor cell proliferation, invasion, and tumor development in multiple cancer models, supporting its role as a potential tumor suppressor.

How does the SNHG22/miR-4492/NKIRAS2 axis regulate cancer progression?

The interaction between NKIRAS2, SNHG22 (small nucleolar RNA host gene 22), and miR-4492 forms a complex regulatory network affecting cancer progression, particularly in osteosarcoma:

• Competing endogenous RNA (ceRNA) mechanism:

  • SNHG22 acts as a ceRNA by directly binding to miR-4492

  • This interaction was validated through RNA pull-down assays, RNA immunoprecipitation, and dual-luciferase reporter assays

  • By sequestering miR-4492, SNHG22 prevents it from targeting NKIRAS2

• Direct targeting of NKIRAS2 by miR-4492:

  • miR-4492 specifically binds to the 3'-UTR of NKIRAS2 mRNA

  • This was confirmed by dual-luciferase reporter assays showing that miR-4492 overexpression impacted NKIRAS2 3'-UTR reporter activity

  • Western blot analysis demonstrated that miR-4492 downregulates NKIRAS2 protein expression while inhibition of miR-4492 upregulates NKIRAS2

• Functional consequences:

  • SNHG22 acts as a protector of NKIRAS2 by sequestering miR-4492

  • miR-4492 promotes OS cell viability and invasion and counteracts the SNHG22-induced tumor-inhibiting effect

  • NKIRAS2 inhibits proliferation and invasion of OS cells

This regulatory axis represents a potential therapeutic target for osteosarcoma and possibly other cancers, where modulation of SNHG22 or inhibition of miR-4492 could restore NKIRAS2 levels and suppress tumor progression.

Through what molecular mechanisms does NKIRAS2 regulate NF-κB signaling?

NKIRAS2 regulates NF-κB signaling through several distinct molecular mechanisms:

• Inhibition of IκB phosphorylation:

  • NKIRAS2 inhibits phosphorylation of IκBβ by the IκB kinase (IKK) complex

  • This inhibition occurs with both GTP- and GDP-bound forms of NKIRAS2

  • By preventing IκBβ phosphorylation, NKIRAS2 protects it from degradation

• Direct interaction with RelA:

  • NKIRAS2 exhibits higher binding affinity with the RelA subunit of the NF-κB complex

  • This interaction inhibits phosphorylation of RelA at Ser-276, which is essential for NF-κB transcriptional activation

  • RelA Ser-276 phosphorylation accumulates in Ras mutant-driven colorectal cancer tissues

• Regulation of NF-κB subcellular localization:

  • NKIRAS2 preferentially binds to IκB-beta through a unique 47-amino acid insert within the ankyrin region

  • While IκB-alpha-NF-κB complexes can shuttle between nucleus and cytoplasm, IκB-beta-NF-κB complexes remain exclusively cytoplasmic

  • By modulating IκB-beta, NKIRAS2 affects NF-κB localization and activity

• Inhibition of RALA small GTPase:

  • NKIRAS2 functions as a tumor suppressor partly through inhibition of RALA small GTPase

  • RALA activates phospholipase D and mTORC1, signaling cascades involved in carcinogenesis

These mechanisms collectively position NKIRAS2 as a multifaceted regulator of NF-κB signaling with implications for inflammation and cancer.

What are the optimal storage and handling conditions for NKIRAS2 antibodies?

To maintain optimal activity of NKIRAS2 antibodies, follow these storage and handling recommendations:

• Storage temperature:

  • Long-term storage: -20°C (stable for up to one year)

  • Short-term storage: 4°C (up to three months)

  • Some formulations may be stored at -80°C for extended preservation

• Formulation considerations:

  • Most commercially available NKIRAS2 antibodies are supplied in:

    • PBS with 0.02-0.09% sodium azide

    • 50% glycerol (pH 7.3) to prevent freeze-thaw damage

    • Some include 0.1M Tris (pH 7), 0.1M Glycine, or 0.01% Thimerosal as preservatives

• Aliquoting and handling:

  • Upon receipt, divide antibody into small working aliquots (10-20 μL)

  • Minimize repeated freeze-thaw cycles which can degrade antibody quality

  • Centrifuge briefly before opening to collect solution at the bottom of the vial

  • Use sterile technique when handling antibody solutions

  • Return to appropriate storage temperature immediately after use

• Stability considerations:

  • At 37°C for 1 month: approximately 80% activity retained

  • At 2-8°C for 6 months: 95-100% activity retained

Proper storage and handling will ensure antibody performance and reproducibility in experimental applications.

What advanced troubleshooting approaches can resolve common issues with NKIRAS2 antibody experiments?

When facing challenges with NKIRAS2 antibody experiments, implement these advanced troubleshooting strategies:

• Western blot inconsistencies:

  • Signal variation between experiments:

    • Standardize lysate preparation (consistent lysis buffer, protease inhibitors)

    • Implement loading controls targeting multiple cellular compartments

    • Consider using total protein normalization (REVERT or similar stains)

  • Unexpected molecular weight detection:

    • While calculated MW is 21.5 kDa, one source reports 68 kDa observations

    • Run gradient gels (4-20%) to better resolve potential isoforms

    • Perform phosphatase treatment to identify phosphorylation-dependent mobility shifts

    • Validate with recombinant NKIRAS2 protein standard as positive control

• Immunofluorescence optimization:

  • Subcellular localization confirmation:

    • Compare multiple fixation methods (4% PFA vs. methanol)

    • Perform co-localization studies with compartment markers (cytoplasmic expected)

    • Consider detergent permeabilization optimization (0.1-0.5% Triton X-100)

  • Signal-to-noise improvement:

    • Implement tyramide signal amplification for weak signals

    • Test alternative blocking reagents (fish gelatin often reduces background)

    • Include 0.1-0.3M glycine to quench aldehyde groups after fixation

• Complex experimental validations:

  • siRNA/shRNA knockdown validation:

    • Design multiple siRNAs targeting different regions of NKIRAS2 mRNA

    • Include rescue experiments with siRNA-resistant NKIRAS2 construct

    • Verify knockdown at both mRNA (qPCR) and protein (Western blot) levels

  • Protein-protein interaction studies:

    • Consider proximity ligation assay (PLA) for endogenous NKIRAS2-NF-κB interactions

    • Use CRISPR-tagged endogenous NKIRAS2 to avoid overexpression artifacts

    • Include appropriate controls for antibody specificity in co-IP experiments

• NKIRAS2 functional assays:

  • Context-dependent effects:

    • Test NKIRAS2 function across multiple cell types (normal vs. cancer)

    • Implement dose-dependent expression systems to evaluate biphasic effects

    • Consider the impact of oncogenic Ras presence on NKIRAS2 function

These advanced troubleshooting approaches address complex technical challenges while enhancing experimental rigor and reproducibility.

How can researchers evaluate NKIRAS2 function in experimental cancer models?

When investigating NKIRAS2 function in cancer models, researchers should consider these methodological approaches:

• Genetic manipulation strategies:

  • Loss-of-function approaches:

    • CRISPR/Cas9-mediated knockout of NKIRAS2

    • shRNA-mediated stable knockdown (multiple shRNA constructs)

    • siRNA-mediated transient knockdown

  • Gain-of-function approaches:

    • Stable overexpression with inducible systems (doxycycline-regulated)

    • Viral delivery (lentiviral/adenoviral) of NKIRAS2 expression constructs

    • Transgenic animal models (tissue-specific expression)

• Functional assays for tumor-related phenotypes:

  • Proliferation assessment:

    • Real-time cell analysis systems (xCELLigence, IncuCyte)

    • Colony formation assays for clonogenic potential

    • Cell cycle analysis by flow cytometry

  • Migration and invasion analysis:

    • Transwell migration and invasion assays

    • Scratch/wound healing assays

    • 3D spheroid invasion assays in extracellular matrix

  • In vivo tumor models:

    • Xenograft models with manipulated NKIRAS2 expression

    • Chemical carcinogenesis models (e.g., DMBA/TPA skin model)

    • Genetic cancer models crossed with NKIRAS2 transgenic lines

• Mechanistic investigation approaches:

  • NF-κB signaling assessment:

    • IκB phosphorylation and degradation kinetics

    • Nuclear translocation of p65/RelA (immunofluorescence)

    • NF-κB transcriptional activity (reporter assays)

  • Pathway crosstalk analysis:

    • NKIRAS2 interactions with Ras signaling components

    • RALA activity monitoring

    • Phospholipase D and mTORC1 signaling evaluation

• Clinical correlation strategies:

  • Expression analysis in human tumor samples:

    • IHC evaluation of NKIRAS2 in patient tumor microarrays

    • Correlation with clinical parameters and survival outcomes

    • Assessment of NKIRAS2 in relation to NF-κB activation markers

These comprehensive approaches enable robust evaluation of NKIRAS2's complex functions in cancer biology.

What experimental evidence explains the paradoxical roles of NKIRAS2 in oncogenic transformation?

NKIRAS2 exhibits context-dependent functions in cellular transformation, with experimental evidence revealing a complex biphasic effect:

• Opposing functions in different cellular contexts:

  • Tumor suppressor function:

    • In osteosarcoma: NKIRAS2 inhibits proliferation and invasion

    • In skin cancer model: K15 promoter-driven NKIRAS2 suppresses DMBA/TPA-induced tumors

  • Transformation-supporting function:

    • In oncogenic HRAS-mutant fibroblasts: NKIRAS2 knockdown suppresses transformation

    • This suggests NKIRAS2 is required for HRAS-driven cellular transformation

• Expression level-dependent biphasic effects:

  • Moderate NKIRAS2 expression:

    • Augments oncogenic HRAS-provoked cellular transformation

    • Potentially supports specific aspects of the transformed phenotype

  • High NKIRAS2 expression:

    • Converts NKIRAS2's role to a tumor suppressive phenotype

    • Creates a bell-shaped response curve where both low and high levels inhibit transformation

• Molecular mechanisms explaining dual roles:

  • NF-κB regulation:

    • Moderate NKIRAS2 may fine-tune NF-κB activity to levels optimal for transformation

    • Excessive NKIRAS2 may completely suppress NF-κB, blocking transformation

  • RALA inhibition:

    • NKIRAS2 inhibits RALA, which is involved in phospholipase D and mTORC1 pathways

    • The balance between RALA inhibition and other NKIRAS2 functions may determine outcomes

• Cell-type specific factors:

  • Differential expression of NKIRAS2 binding partners

  • Varying baseline NF-κB activity levels

  • Presence of other oncogenic drivers or tumor suppressors

This complex behavior suggests that NKIRAS2-targeted therapeutic approaches would require precise contextual understanding and possibly dosage control to achieve desired outcomes in cancer treatment.

What critical validation steps ensure specificity and reliability of NKIRAS2 antibodies?

Rigorous validation of NKIRAS2 antibodies requires multiple complementary approaches:

• Primary specificity validation:

  • Genetic knockdown/knockout controls:

    • siRNA/shRNA-mediated NKIRAS2 knockdown

    • CRISPR/Cas9-mediated NKIRAS2 knockout

    • Confirmation of signal reduction in Western blot and immunostaining

  • Epitope blocking experiments:

    • Pre-incubation with immunizing peptide should abolish specific signal

    • Western blot comparison with/without blocking peptide

    • Use in both Western blot and immunohistochemistry applications

• Cross-reactivity assessment:

  • Multi-species reactivity testing:

    • Human NKIRAS2 is the primary target for most antibodies

    • Cross-reactivity with mouse and rat NKIRAS2 should be experimentally confirmed

    • Sequence alignment analysis to predict reactivity with other species

  • Family member discrimination:

    • Verification of specificity against NKIRAS1, the closest homolog

    • Testing in cells with differential expression of NKIRAS1 vs NKIRAS2

    • Epitope mapping to confirm targeting of unique regions

• Technical performance validation:

  • Application-specific assessment:

    • Western blot: Detection of expected 21-22 kDa band

    • Immunofluorescence: Expected cytoplasmic localization

    • Immunoprecipitation: Efficient pulldown from cell lysates

  • Multi-antibody concordance:

    • Comparison of results using antibodies targeting different NKIRAS2 epitopes

    • Correlation of protein detection with mRNA expression data

    • Commercial vs. custom antibody performance comparison

• Recombinant protein standards:

  • Use of purified recombinant NKIRAS2:

    • Titration curves to determine detection limits

    • Confirmation of expected molecular weight

    • Comparison to endogenous protein detection patterns

Implementation of these validation steps ensures that experimental results with NKIRAS2 antibodies are specific, reproducible, and biologically relevant.

What are the promising research directions for NKIRAS2 in cancer and inflammatory diseases?

Based on current understanding of NKIRAS2 biology, several promising research avenues are emerging:

• Therapeutic targeting strategies:

  • Small molecule modulators:

    • Development of compounds that enhance NKIRAS2 stability or activity in cancers where it acts as a tumor suppressor

    • Context-specific inhibitors for scenarios where NKIRAS2 supports oncogenic transformation

    • Structure-based drug design targeting NKIRAS2-RelA or NKIRAS2-IκB interfaces

  • miRNA-based approaches:

    • Anti-miR-4492 therapies to relieve suppression of NKIRAS2

    • Development of SNHG22 mimetics to sequester miR-4492

    • Targeted delivery systems for these nucleic acid therapeutics

• Biomarker development:

  • Prognostic indicators:

    • NKIRAS2 expression levels as predictors of cancer progression

    • SNHG22/miR-4492/NKIRAS2 axis evaluation in patient samples

    • Correlation with response to conventional therapies

  • Therapeutic response markers:

    • NKIRAS2 as a predictor of response to NF-κB-targeting therapies

    • Expression patterns in therapy-resistant vs sensitive tumors

    • Liquid biopsy approaches to monitor NKIRAS2 pathway status

• Expansion to additional disease contexts:

  • Inflammatory disorders:

    • NKIRAS2 function in chronic inflammatory conditions

    • Role in autoimmune disease pathogenesis

    • Potential as an anti-inflammatory therapeutic target

  • Additional cancer types:

    • Beyond osteosarcoma and skin cancer

    • Hematological malignancies where NF-κB is crucial

    • Context-dependent roles across cancer subtypes

• Advanced mechanistic investigations:

  • Structural biology:

    • Crystal structure of NKIRAS2 in complex with IκB and/or RelA

    • Conformational changes associated with GTP/GDP binding

    • Molecular basis for biphasic effects in cellular transformation

  • Systems biology:

    • Integration of NKIRAS2 into comprehensive NF-κB regulatory networks

    • Mathematical modeling of dose-dependent effects

    • Multi-omics approaches to define NKIRAS2-dependent cellular states

These research directions promise to expand our understanding of NKIRAS2 biology and potentially reveal new therapeutic opportunities for cancer and inflammatory diseases.