NEK9 Antibody

What is NEK9 and what are its key functional domains?

NEK9 is a NEK-type protein kinase that regulates chromosome alignment and segregation during mitosis. It features three main structural domains:

• N-terminal NIMA-like catalytic domain

• Central domain with homology to RCC1 (regulator of chromosome condensation 1)

• C-terminal coiled-coil domain

NEK9 undergoes phosphorylation by active p34(Cdc2), exhibits autophosphorylation capabilities, and forms oligomers when activated. The activated protein interacts with Nek6, RCC1, and has been shown to associate with Bicaudal D (Bicd2) in vivo, phosphorylating this coiled-coil protein in vitro .

Which NEK9 antibodies are commonly used for basic research applications?

Several NEK9 antibodies have been developed for research purposes:

For most basic research applications like protein detection and localization studies, both polyclonal and monoclonal antibodies work well, with monoclonals providing better specificity for particular epitopes .

What experimental methods can verify NEK9 antibody specificity?

To ensure NEK9 antibody specificity:

• Western blot validation with knockout controls: Compare wild-type versus NEK9 knockout cell lysates to confirm band specificity (observed band size ~120 kDa versus predicted 107 kDa) .

• Peptide competition assay: Pre-incubate the antibody with immunizing peptide before immunoblotting or immunostaining to verify signal elimination .

• Phospho-specificity tests: For phospho-antibodies, compare untreated samples with samples treated with phosphatase or stimuli known to induce phosphorylation .

• Cross-reactivity assessment: Test on multiple species samples (human, mouse, rat) to confirm conservation of recognized epitopes .

• Multiple detection methods: Confirm protein identity using two or more antibodies targeting different NEK9 epitopes .

How should NEK9 antibodies be optimized for Western blotting?

For optimal Western blot results with NEK9 antibodies:

• Sample preparation: NEK9 is a large protein (107 kDa) that typically runs at ~120-130 kDa on gels due to post-translational modifications. Use 15-30 μg of total protein lysate per well .

• Gel separation: Use 6-8% SDS-PAGE gels or 4-12% gradient gels to achieve proper separation of high molecular weight proteins.

• Transfer conditions: Employ wet transfer at lower amperage (e.g., 30V overnight at 4°C) to ensure complete transfer of larger proteins.

• Antibody concentration: For most NEK9 antibodies, a 1:500-1:1000 dilution works well for primary antibody incubation .

• Blocking conditions: 5% non-fat dry milk in TBST is typically effective, though some phospho-specific antibodies perform better with 5% BSA .

• Controls: Include positive controls (e.g., HeLa, HepG2, or MCF7 cell lysates) which express detectable NEK9 levels .

• Expected result: A primary band at approximately 120 kDa, with potential minor bands representing isoforms or degradation products .

What are the best protocols for immunofluorescence with NEK9 antibodies?

For successful immunofluorescence detection of NEK9:

• Fixation: Methanol fixation (100% methanol at -20°C for 10 minutes) preserves NEK9 epitopes better than paraformaldehyde for many antibodies .

• Permeabilization: If using paraformaldehyde fixation, permeabilize with 0.1% Triton X-100 for 5-10 minutes .

• Antibody dilution: Use 1:100-1:200 dilution of primary NEK9 antibody in blocking buffer .

• Incubation: Overnight incubation at 4°C yields the best signal-to-noise ratio .

• Detection: Use appropriate secondary antibodies conjugated to fluorophores like Alexa Fluor 488, 555, or 647 .

• Co-staining: For mitosis studies, co-stain with γ-tubulin for centrosomes; for cilia studies, co-stain with acetylated tubulin .

• Expected localization: NEK9 typically shows cytoplasmic distribution with enrichment at centrosomes during mitosis; a small portion localizes to the nucleus .

How can NEK9 phosphorylation status be monitored using phospho-specific antibodies?

Monitoring NEK9 phosphorylation:

• Available phospho-antibodies: Commercially available antibodies target phospho-Thr210 (activation loop) and phospho-Ser869 (Plk1 binding site) .

• Cell cycle synchronization: For mitotic phosphorylation, synchronize cells using nocodazole (100 ng/ml for 16 hours) or thymidine-nocodazole block .

• Inhibitor studies: Use CDK1 inhibitors (e.g., RO-3306) or Plk1 inhibitors (e.g., BI2536) to block specific phosphorylation events .

• Mutation studies: Compare wild-type NEK9 with phospho-site mutants (e.g., T210A, S869A) to confirm antibody specificity .

• Western blot detection: Phosphorylated NEK9 often appears as a mobility-shifted band (higher molecular weight) .

• Immunofluorescence patterns: Phosphorylated NEK9 shows distinct localization patterns, with phospho-T210 NEK9 enriched at centrosomes during prophase .

• Quantification: Use densitometry of immunoblots or fluorescence intensity measurements in immunostained cells to quantify phosphorylation levels .

How can NEK9 antibodies be used to study cell cycle regulation and mitotic progression?

NEK9 plays a crucial role in mitotic progression, particularly in centrosome separation:

• Immunoprecipitation studies: Use NEK9 antibodies to pull down protein complexes and identify cell-cycle-specific interaction partners. Key protocols include:

  • Crosslinking antibodies to beads (protein A/G) to reduce background

  • Using phosphatase inhibitors in lysis buffers to preserve phosphorylation-dependent interactions

  • Sequential immunoprecipitation to identify transient complexes

• Cell synchronization and time-course analysis:

  • For S-phase: Double thymidine block

  • For mitotic entry: Thymidine-nocodazole block

  • For mitotic exit: Nocodazole arrest and release

  • Collect samples at 2-hour intervals for western blot analysis of NEK9 and its phosphorylation

• Centrosome separation assays:

  • Treat cells with NEK9 siRNA for 48-72 hours

  • Fix cells and stain with antibodies against γ-tubulin (centrosomes) and NEK9

  • Categorize cells based on centrosome separation (unseparated, partially separated, fully separated)

  • Rescue experiments by expressing siRNA-resistant wild-type or mutant NEK9

• Mitotic kinase assays:

  • Immunoprecipitate NEK9 from mitotic cells

  • Assay kinase activity using histone H3 or specific substrates

  • Compare activity with or without specific inhibitors

  • Use phospho-specific antibodies to detect substrate phosphorylation

What are the best approaches for studying NEK9's role in cancer cells lacking functional p53?

NEK9 plays a critical role in cancer cells lacking functional p53, offering therapeutic targeting opportunities:

• Expression correlation analysis:

  • Compare NEK9 levels between p53-wildtype and p53-mutant cell lines using western blotting

  • Quantify NEK9 expression in patient samples using immunohistochemistry

  • Correlate NEK9 expression with patient survival data

• Functional studies in p53-deficient versus p53-wildtype contexts:

  • Knockdown NEK9 using siRNA in matched cell lines with different p53 status

  • Measure effects on proliferation, colony formation, and cell cycle progression

  • Rescue experiments using NEK9 expression constructs to confirm specificity

• Cell cycle analysis in p53-deficient cells:

  • Synchronize cells and analyze cell cycle profiles by flow cytometry after NEK9 knockdown

  • Determine cell cycle phase arrest (primarily G1) following NEK9 depletion

  • Assess senescence markers (β-galactosidase activity, p21 expression)

• Gene expression profiling:

  • Perform RNA-seq or microarray analysis after NEK9 knockdown in p53-deficient cells

  • Identify downstream pathways affected by NEK9 depletion

  • Focus on cell cycle regulators and mRNA processing factors

• In vivo tumor growth studies:

  • Generate xenografts with p53-deficient cancer cells with or without NEK9 knockdown

  • Monitor tumor growth and analyze tumor tissues for proliferation markers

  • Correlate findings with patient data from cancer genomics databases

How can NEK9 antibodies be used to investigate its role in viral infections like adenovirus?

NEK9 plays a dual role during adenovirus infection, affecting viral gene expression and genome replication:

• Virus-host protein interaction studies:

  • Use co-immunoprecipitation with NEK9 antibodies to isolate viral protein complexes

  • Identify viral proteins (e.g., E1A, E4 orf3) that interact with NEK9

  • Perform reciprocal IPs using viral protein antibodies to confirm interactions

• Subcellular localization during infection:

  • Track NEK9 redistribution using immunofluorescence at various time points post-infection

  • Co-stain with viral replication centers using antibodies against viral DNA binding protein (DBP)

  • Analyze nuclear vs. cytoplasmic fractions by western blotting

• Impact on viral replication:

  • Knockdown NEK9 using siRNA prior to infection

  • Measure viral genome copies using qPCR

  • Analyze expression of viral genes from different transcription units (E1A, E2, E3, E4)

  • Determine virus titers using plaque assays

• Chromatin immunoprecipitation (ChIP):

  • Use NEK9 antibodies for ChIP to detect association with viral promoters

  • Analyze binding to cellular stress response gene promoters (e.g., GADD45A)

  • Compare binding patterns between infected and uninfected cells

• NEK9 knockdown/overexpression effects:

  • Establish stable cell lines with inducible NEK9 expression or knockdown

  • Infect with adenovirus and measure viral gene expression, protein levels, and replication

  • Rescue experiments with wild-type or mutant NEK9 to identify functional domains

What methods can be used to study NEK9's role in primary cilia formation?

NEK9 regulates primary cilia formation by acting as a selective autophagy adaptor for MYH9:

• Ciliogenesis assays:

  • Induce cilia formation by serum starvation (24-48 hours)

  • Stain for cilia markers (acetylated tubulin, ARL13B) and NEK9

  • Quantify cilia length and formation frequency in control versus NEK9-depleted cells

• NEK9-ATG8 interaction studies:

  • Perform co-IP experiments with NEK9 antibodies to detect interaction with LC3 or GABARAP proteins

  • Use NEK9 mutants (W967A) with disrupted LC3-interacting region (LIR) to confirm specificity

  • Analyze localization of NEK9 to autophagosomes using fluorescence microscopy

• NEK9-MYH9 regulation:

  • Measure MYH9 (myosin IIA) levels in wild-type versus NEK9 knockout or LIR mutant cells

  • Perform rescue experiments with MYH9 knockdown in NEK9 LIR mutant cells

  • Analyze actin network dynamics in relation to ciliogenesis

• In vivo cilia formation:

  • Generate NEK9 LIR mutant mice (W967A)

  • Analyze kidney sections for cilia formation using immunofluorescence

  • Compare cilia length and frequency between wild-type and mutant tissues

• Autophagy flux assays:

  • Measure selective autophagy of MYH9 using bafilomycin A1 treatment

  • Compare autophagy markers (LC3-II, p62) in wild-type versus NEK9 mutant cells

  • Analyze MYH9 turnover rates with cycloheximide chase experiments

What are common issues with NEK9 detection in Western blots and how can they be resolved?

Common Western blot issues and solutions:

• Multiple bands or non-specific binding:

  • Increase blocking time (2 hours at room temperature or overnight at 4°C)

  • Try different blocking agents (milk vs. BSA vs. commercial blockers)

  • Increase washing steps (5× 5 minutes with TBST)

  • Titrate antibody concentration to determine optimal dilution

• Weak or no signal:

  • Confirm NEK9 expression in your cell type (higher in liver, heart, kidney, testis)

  • Increase protein loading (up to 50 μg)

  • Lengthen exposure time or use more sensitive detection methods

  • Check transfer efficiency with reversible protein stains

• Unexpected molecular weight:

  • NEK9 typically runs at ~120-130 kDa despite a predicted size of 107 kDa due to post-translational modifications

  • Phosphorylated forms may appear as mobility-shifted bands

  • Alternative splicing can produce variant forms

• Degradation products:

  • Use fresh samples and maintain cold temperatures during extraction

  • Add protease inhibitors to lysis buffer

  • Avoid repeated freeze-thaw cycles of samples

• Phospho-specific antibody issues:

  • Use phosphatase inhibitors during extraction (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Block with BSA instead of milk for phospho-antibodies

  • Consider lambda phosphatase treatment as a negative control

How should experiments be designed to investigate NEK9 phosphorylation dynamics?

Designing experiments to investigate NEK9 phosphorylation:

• Cell cycle synchronization protocols:

  • G1/S arrest: Double thymidine block (2 mM thymidine for 16 hours, release for 8 hours, second block for 16 hours)

  • Mitotic arrest: Nocodazole treatment (100 ng/ml for 16 hours)

  • Sequential collection during cell cycle progression (every 2 hours after release)

• Kinase inhibitor treatments:

  • CDK1 inhibition: RO-3306 (10 μM)

  • Plk1 inhibition: BI2536 (100 nM)

  • Determine timing and concentration through dose-response and time-course experiments

• Phospho-site mutant construction:

  • Generate site-specific mutants (S→A or T→A) using site-directed mutagenesis

  • Create phosphomimetic mutants (S→D or T→E) to study constitutive activation

  • Express in cells using appropriate vectors (transient vs. stable expression)

• Mass spectrometry analysis:

  • Immunoprecipitate NEK9 from synchronized cells

  • Perform in-gel digestion with trypsin

  • Analyze phosphopeptides using LC-MS/MS

  • Compare phosphorylation patterns across cell cycle stages

• Antibody validation for phospho-specificity:

  • Compare phospho-antibody reactivity before and after phosphatase treatment

  • Test antibody reactivity against phospho-site mutant proteins

  • Use competing phosphopeptides to confirm specificity

What controls should be used when studying NEK9 using antibody-based techniques?

Essential controls for NEK9 antibody experiments:

• Positive controls:

  • Cell lines with known NEK9 expression (HeLa, HepG2, MCF7)

  • Tissue samples with high NEK9 expression (liver, kidney, testis)

  • Recombinant NEK9 protein (for Western blot standardization)

• Negative controls:

  • NEK9 knockout or knockdown cells (using CRISPR-Cas9 or siRNA)

  • Secondary antibody only (to detect non-specific binding)

  • Irrelevant primary antibody of same isotype

• Specificity controls:

  • Peptide competition assays (pre-incubate antibody with immunizing peptide)

  • Multiple antibodies targeting different NEK9 epitopes

  • Test for cross-reactivity with other NEK family members

• Phosphorylation controls:

  • Lambda phosphatase treatment (to remove phosphate groups)

  • Cells treated with kinase inhibitors (CDK1, Plk1)

  • Phospho-site mutant expression (S→A or T→A)

• Loading controls:

  • GAPDH or tubulin for whole cell lysates

  • Lamin B for nuclear fractions

  • Normalize NEK9 signal to loading control for quantitative comparisons

• Cell-Based ELISA normalization controls:

  • Use GAPDH normalization for relative quantification

  • Employ crystal violet staining for cell number normalization

  • Calculate normalized values using the proportion OD450/OD595

How should quantitative data from NEK9 experiments be analyzed and presented?

Best practices for NEK9 data analysis:

• Western blot quantification:

  • Use digital imaging and densitometry software (ImageJ, Image Lab)

  • Normalize NEK9 signal to appropriate loading control

  • Present data as fold-change relative to control conditions

  • Include representative blot images alongside quantification graphs

• Immunofluorescence quantification:

  • Analyze at least 100-200 cells per condition

  • For centrosome separation: categorize as unseparated (<2 μm), partially separated (2-5 μm), or fully separated (>5 μm)

  • For cilia studies: measure percentage of ciliated cells and cilia length

  • Use box plots or violin plots for distribution data

• Cell proliferation assays:

  • Present growth curves over multiple time points (24, 48, 72 hours)

  • Normalize to control conditions at each time point

  • Calculate doubling times and growth rates

  • Use appropriate statistical tests (t-test or ANOVA with post-hoc tests)

• Gene expression data:

  • Normalize to appropriate reference genes

  • Use fold-change and statistical significance (p-value) cutoffs

  • Perform pathway enrichment analysis

  • Visualize with heatmaps, volcano plots, or enrichment plots

• Statistical analysis recommendations:

  • Perform experiments in triplicate (minimum) with biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Report mean ± standard deviation or standard error

  • Indicate p-values and significance thresholds

How do NEK9 functions differ across cell types and physiological contexts?

NEK9 shows context-dependent functions across different systems:

• Cell cycle regulation:

  • In proliferating cells: Essential for prophase centrosome separation via Plk1-dependent activation

  • In mitotic cells: Activates downstream NEK6/7 kinases to regulate Eg5 motor protein

  • In interphase cells: Required for G1/S transition and S phase progression

• Cancer context:

  • In p53-mutant cancers: Critical for proliferation, cell cycle progression

  • In p53-wildtype cells: Less essential for growth

  • Expression levels correlate with poor prognosis in lung adenocarcinoma when co-expressed with mutant p53

• Primary cilia regulation:

  • In quiescent cells: Required for ciliogenesis via autophagic degradation of MYH9

  • In NEK9 LIR mutants: Impaired cilia formation due to MYH9 accumulation

  • Tissue-specific effects observed in kidney epithelial cells

• Viral infection response:

  • During adenovirus infection: Acts as a transcriptional repressor of stress response genes

  • Undergoes subcellular redistribution in response to viral E1A protein

  • Affects viral genome replication in complex ways

• Tissue expression patterns:

  • High expression in normal brain, bone, lymph, thymus

  • Elevated expression in cancerous tissues (cervix, eye, lymph, placenta)

  • Expression in lung cancer subtypes: 57.9% of adenocarcinomas, 96.4% of squamous cell carcinomas, 100% of large cell neuroendocrine and small cell carcinomas

What are the latest research directions and emerging applications for NEK9 antibodies?

Cutting-edge NEK9 research applications:

• Therapeutic targeting in p53-deficient cancers:

  • Using NEK9 antibodies for patient stratification in clinical trials

  • Developing NEK9 inhibitors as synthetic lethal therapies for p53-mutant cancers

  • Identifying NEK9-dependent vulnerabilities through proteomics and CRISPR screens

• Cilia-related disease research:

  • Investigating NEK9's role in ciliopathies using patient-derived cells

  • Exploring the NEK9-LIR-autophagy axis in polycystic kidney disease models

  • Developing tools to monitor selective autophagy of MYH9 mediated by NEK9

• Systems biology approaches:

  • Mapping the NEK9 protein interactome across cell cycle phases

  • Integrating phosphoproteomic and transcriptomic data to build NEK9 signaling networks

  • Using computational modeling to predict context-dependent NEK9 functions

• High-content imaging applications:

  • Automated analysis of NEK9 localization dynamics during mitosis

  • Quantitative phenotyping of cellular responses to NEK9 modulation

  • Live-cell imaging with fluorescently tagged NEK9 to track real-time dynamics

• Novel antibody-based technologies:

  • Proximity labeling methods (BioID, APEX) using NEK9 antibodies to map local interactomes

  • Nanobodies or intrabodies targeting specific NEK9 conformations or complexes

  • Antibody-drug conjugates targeting NEK9-expressing cancer cells