CEP131 Antibody

What is CEP131 and what cellular functions does it regulate?

CEP131 is an evolutionarily conserved centriolar satellite protein that contributes to building a complex network regulating cilia/flagellum formation . It plays multiple roles in cellular processes including:

• Maintenance of genome stability

• Cell cycle progression and proliferation

• Centriole duplication

• Centrosomal protein localization (for CEP152, WDR62, and CEP63)

• Regulation of BBSome ciliary trafficking

In proliferating cells, MIB1-mediated ubiquitination induces its sequestration within centriolar satellites, preventing premature cilia formation. During normal or stress-induced ciliogenesis, non-ubiquitinated CEP131 relocates to centrosome/basal bodies in a microtubule and p38 MAPK-dependent manner .

What is the subcellular localization pattern of CEP131?

CEP131 shows distinct localization patterns that change according to cellular state:

• Normal proliferating cells: Primarily localized to centriolar satellites with some centrosomal presence

• During ciliogenesis: Redistributed from satellites to centrosomes/basal bodies

• Cancer tissues: Often shows both nuclear and cytoplasmic localization

• Tissue-specific: In testis, CEP131 localizes to the pre-acrosome region of round and elongated spermatids

When using immunofluorescence, proper fixation methods are critical as they can affect satellite integrity. For optimal visualization of CEP131 at centriolar satellites, methanol fixation is generally recommended.

What experimental approaches can detect changes in CEP131 expression?

Based on published research, the following techniques have been validated for CEP131 expression analysis:

When conducting these assays, appropriate controls and validation of antibody specificity are essential for reliable results.

How can researchers experimentally manipulate CEP131 expression for functional studies?

Several approaches have been validated in the literature:

siRNA-mediated knockdown:

• Used successfully in A549 and SPC-A-1 lung cancer cell lines

• Pooled siRNA targeting approach recommended with at least 48h post-transfection for protein reduction

• Western blotting showed substantial reduction in CEP131 protein levels following 48h of siRNA treatment

• siRNA targeting 3′ UTR can be used for rescue experiments with exogenous CEP131 expression

CRISPR-Cas9 knockout:

• Complete deletion achievable in cell lines and animal models

• Interestingly, acute versus chronic loss produces different phenotypes in ciliogenesis

• Can target C-terminal regions as demonstrated in Drosophila models

Overexpression systems:

• Lentiviral-mediated expression of V5-tagged CEP131 demonstrated in neuroblastoma cells

• Proper localization of exogenous protein should be verified via co-localization with endogenous CEP131

What are the emerging connections between CEP131 and cancer progression?

CEP131 has been implicated in multiple cancer types with emerging evidence for mechanistic roles:

Non-small cell lung cancer (NSCLC):

• High expression in 63.7% (58/91) of NSCLC cases

• Significantly associated with advanced TNM stage (P=0.016) and positive lymph node metastasis (P=0.023)

• Expression breakdown by histological type: 65.9% (27/41) of squamous cell carcinoma cases and 62.0% (31/50) of adenocarcinoma cases

Neuroblastoma:

Other cancers:

• Oncogenic activity reported in osteosarcoma, hepatocellular carcinoma, and breast cancer

• Higher expression correlates with higher histologic grades in breast cancer

How does CEP131 impact intracellular signaling pathways?

CEP131 influences multiple signaling networks, with knockdown studies revealing specific pathway dysregulation:

ERK and PI3K/AKT pathways:
In CEP131-knockdown A549 and SPC-A-1 cells, significant decreases were observed in:

• p-PI3K (Tyr458): reduced to 0.51±0.11 and 0.52±0.08 of control levels

• p-AKT (Ser473): reduced to 0.60±0.09 and 0.58±0.12

• p-MEK1/2 (Ser-217/221): reduced to 0.55±0.04 and 0.70±0.03

• p-ERK1/2 (Tyr202/Tyr204): reduced to 0.58±0.24 and 0.68±0.16

• p-GSK-3β (Ser-9): reduced to 0.57±0.18 and 0.63±0.09

These changes were associated with downstream effects on cell cycle regulators, including:

• Reduced expression of cyclins D1/E and CDKs 2/4/6

• Increased expression of cell cycle inhibitors p21/p27

T-cell receptor signaling:

• CEP131 participates in the maintenance of proximal TCR components

• Affects CD3ε trafficking and subsequent signaling

What technical considerations are important when using CEP131 antibodies for immunoprecipitation studies?

For successful immunoprecipitation (IP) experiments with CEP131:

• Lysis conditions:

  • Use buffers containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton X-100, 1 mM dithiothreitol, 10% glycerol, and protease inhibitors

  • Sonication may help solubilize centriolar satellite components

• Controls for IP specificity:

  • Always include IgG control IP to detect non-specific binding

  • Validate IP efficiency by immunoblotting input, unbound, and bound fractions

  • In co-IP experiments, include reciprocal IP when possible

• Detection of protein interactions:

  • The interaction between CEP131 and MDM2 was successfully detected in neuroblastoma cells treated with CHK1 inhibitor

  • For purified protein interaction assays, recombinant His-tagged CEP131 truncations can be used with immobilized GST-tagged potential interactors

• Caspase cleavage considerations:

  • CEP131 can be cleaved by CASP3 after aspartic acid in position 548

  • When studying apoptosis, consider using caspase inhibitors if full-length CEP131 is needed

What methodological approaches can distinguish between acute and chronic CEP131 loss phenotypes?

Research has revealed important differences between acute and chronic loss of CEP131 function:

Acute depletion (siRNA knockdown):

• Results in reduced cilia formation in mammalian cells

• Affects cell cycle progression and proliferation

• Causes instability in cilia network

Chronic deletion (genetic knockout):

• In mice, cilia form normally despite complete absence of Azi1/CEP131

• Mice develop normally but display male infertility due to defects in sperm flagellar formation

• System appears to re-equilibrate, allowing cilia to form through compensatory mechanisms

To properly examine these differences, researchers should consider:

• Using both transient (siRNA) and stable (CRISPR) approaches in parallel

• Implementing rescue experiments with wild-type or mutant CEP131

• Examining short-term versus long-term phenotypes in the same experimental system

• Monitoring potential compensatory mechanisms through proteomic analysis

How does CEP131 influence microtubule dynamics and post-translational modifications?

CEP131 has emerged as a regulator of microtubule properties:

• Microtubule regrowth:

  • Cells lacking CEP131 show reduction in microtubule regrowth after nocodazole-induced depolymerization

  • This suggests a role in microtubule nucleation or stabilization

• Tubulin post-translational modifications:

  • CEP131 deletion reduces glycylated and polyglutamylated tubulin species

  • No significant effect observed on tubulin detyrosination

  • These modifications comprise the "tubulin code" that regulates microtubule dynamics and interactions with motor proteins and MAPs

• Methodological approach to study these effects:

  • Nocodazole washout assay with timed recovery periods

  • Immunofluorescence using modification-specific antibodies

  • Western blotting to quantify relative levels of modified tubulin

What is the role of CEP131 in mitochondrial functions and apoptosis?

Recent research has uncovered unexpected roles for CEP131 in mitochondrial biology:

• Mitochondrial morphology:

  • CEP131-deficient cells show elongated mitochondrial networks

  • No significant changes in mitochondrial mass (as measured by MitoTracker Green) or mitochondrial DNA content

• Mitochondrial membrane potential:

  • CEP131 knockout cells exhibit increased mitochondrial membrane potential (measured by TMRM fluorescence)

• Apoptotic resistance:

  • CEP131-deficient cells show delayed cytochrome c release from mitochondria

  • Subsequent caspase activation and apoptosis progression is delayed

  • This mitochondrial permeabilization defect is intrinsic and replicable with isolated organelles

• Experimental approach:

  • Flow cytometry with MitoTracker Green for mass determination

  • TMRM for membrane potential assessment

  • Structure illumination microscopy (SIM) for detailed morphological analysis

  • Isolated mitochondria experiments for direct assessment of permeabilization

What mechanisms regulate CEP131 protein stability and degradation?

CEP131 levels are tightly controlled through multiple mechanisms:

• Ubiquitination:

  • MIB1 E3 ligase mediates ubiquitination of CEP131

  • This modification affects its subcellular localization and sequestration at centriolar satellites

• MDM2-mediated degradation:

  • In neuroblastoma cells, CHK1 inhibitor (PF-477736) treatment leads to:

    • Increased MDM2 expression

    • Destabilization of CEP131 protein

  • siRNA-mediated depletion of MDM2 reverses CEP131 destabilization despite CHK1 inhibition

  • Cycloheximide chase experiments demonstrated accelerated CEP131 degradation in the presence of CHK1 inhibitor

• Caspase cleavage:

  • CASP3 directly cleaves CEP131 after aspartic acid in position 548

  • This cleavage occurs during apoptosis and contributes to centriolar satellite remodeling

How does CEP131 regulate cell cycle progression in normal and cancer cells?

CEP131 plays important roles in cell cycle control, particularly at the G1/S transition:

• Cell cycle phase distribution:

  • CEP131 knockdown in A549 and SPC-A-1 cells significantly:

    • Increases the percentage of cells in G1 phase

    • Decreases the percentage in S phase

    • No significant changes detected in G2 phase

• Cell cycle-related protein regulation:

  • CEP131 downregulation reduces expression of:

    • Cyclin D1 and cyclin E (G1 and G1/S phase cyclins)

    • CDK2, CDK4, and CDK6 (G1/S phase kinases)

  • CEP131 downregulation increases expression of:

    • p21 and p27 (CDK inhibitors that block G1/S transition)

• Upstream signaling pathways:

  • CEP131 regulates cell cycle via the ERK and PI3K/Akt pathways

  • GSK-3β, a downstream regulator of these pathways, is affected by CEP131 knockdown

  • Inhibition of GSK-3β through phosphorylation normally induces G1 to S phase transition

These findings suggest CEP131 promotes cell proliferation by facilitating G1/S transition through multiple interconnected mechanisms.

What experimental approaches can determine if CEP131 variants affect centriole duplication?

To investigate CEP131's role in centriole duplication:

• Quantitative immunofluorescence:

  • Stain for centriole markers (e.g., centrin, CP110) in CEP131-depleted cells

  • Count centriole numbers per cell across cell cycle phases

  • Use cell cycle markers to determine specific effects

• Live cell imaging:

  • Generate stable cell lines expressing fluorescently-tagged centriole markers

  • Perform time-lapse imaging through multiple cell divisions

  • Analyze timing and efficiency of centriole duplication

• Rescue experiments:

  • Test different CEP131 domains/truncations for ability to restore normal centriole duplication

  • Particularly examine the N-terminal (1-549 aa) and C-terminal (550-1114 aa) domains

  • Identify specific mutations that affect this function

• Electron microscopy:

  • Ultrastructural analysis to detect subtle defects in centriole structure

  • Examine cartwheel assembly in early duplication stages

Previous research has noted that knockdown of CEP131 can lead to a slight increase in cells with extra centrioles , suggesting its role in maintaining normal centriole number.