CBY1 Antibody

• How can I distinguish between different CBY proteins (CBY1, CBY3) in my experiments?

Distinguishing between CBY family members requires careful antibody selection and experimental design:

• Epitope specificity: Ensure your selected antibody specifically recognizes CBY1 rather than other family members. Many commercial antibodies are developed against full-length CBY1 or specific regions (e.g., AA 1-126, AA 41-126) .

• Expression pattern analysis: CBY1 is widely expressed across tissues, while CBY3 is exclusively expressed in testis . When working with testicular tissue, carefully validate antibody specificity.

• Molecular weight differences: Although both proteins are similar in size, they may show subtle differences in migration patterns during Western blotting.

• For advanced discrimination, consider:

  • Using multiple antibodies targeting different epitopes

  • Performing knockdown/knockout validation experiments

  • Using recombinant proteins as positive controls

  • Employing mass spectrometry for definitive identification

When studying CBY3, note that it interacts with ciBAR1 at the annulus in differentiating spermatids through specific residues (S5, T6, E18, G20, Y23, and R29), forming complexes similar to CBY1/ciBAR1 complexes .

• How do I troubleshoot inconsistent CBY1 antibody staining in immunofluorescence experiments?

Inconsistent CBY1 staining can result from several factors:

• Subcellular localization variations: CBY1 localizes to multiple cellular compartments including:

  • Golgi apparatus (specifically trans-Golgi network)

  • Cilium basal body

  • Centrioles

  • Nuclear speckles

  This diverse localization can appear as inconsistent staining if fixation conditions preferentially preserve certain compartments.

• Fixation-dependent epitope masking: Try different fixation methods:

  • 4% PFA (overnight at 4°C) for tissue samples

  • 100% acetone (10 min at -20°C) for frozen sections

  • Methanol fixation may better preserve certain epitopes

• Extraction requirements: CBY1 detection often requires membrane permeabilization:

  • Use 0.5% Triton X-100 in PBS (5 min) or 0.5% SDS in PBS (10 min)

  • For sperm samples, careful extraction with 0.5% Triton X-100 after fixation

• Signal amplification: For low abundance detection, implement tyramide signal amplification using kits such as the Alexa Fluor 488 Tyramide SuperBoost Kit

• Cell-cycle dependence: CBY1 localization changes throughout the cell cycle, especially at centrosomes/cilia. Synchronize cells if examining cell-cycle-dependent localization.

• How can I interpret CBY1 expression changes in the context of epithelial-mesenchymal transition (EMT) studies?

Interpreting CBY1 expression changes in EMT studies requires careful consideration of several factors:

• Temporal dynamics: CBY1 knockdown induces mesenchymal-to-epithelial transition (MET)-like changes, with acute versus chronic knockdown producing different effects on Wnt signaling . When analyzing EMT/MET:

  • Document the duration of CBY1 modulation

  • Consider both short-term and long-term effects

• Cell-context dependency: CBY1 knockdown effects are consistent across different cell types (SW480 colon cancer cells and HEK293 cells), but the magnitude varies:

  • HEK293 cells show more dramatic morphological changes than SW480 cells

  • Colony formation reduction is more pronounced in HEK293 cells (97%) than in SW480 cells (48%)

• Marker panel analysis: CBY1 knockdown triggers a complex set of cellular changes affecting multiple markers:

  Marker	Effect of CBY1 Knockdown	Significance
  E-cadherin	5-fold protein increase, 2.4-fold mRNA increase	Adherens junction formation
  β-catenin	Significant increase at adherens junctions	Cell-cell adhesion enhancement
  ZO-1	6-fold protein increase	Tight junction formation
  Vimentin	Decreased levels	Reduced mesenchymal properties
  Actin	Formation of cortical rings	Epithelial cytoskeletal arrangement
  TopFlash (Wnt activity)	Decreased in stable CBY1-KD SW480 cells	Reduced β-catenin signaling

• Migration pattern analysis: CBY1 knockdown cells maintain epithelial characteristics during migration, moving as coherent sheets rather than individual cells . This can help distinguish true EMT/MET processes from other cellular changes.

• What are the best approaches for validating CBY1 antibody specificity in experimental systems?

Comprehensive validation of CBY1 antibody specificity should include:

• Genetic models:

  • Use CBY1 knockout (KO) cells/tissues as negative controls

  • Compare staining patterns in CBY1-depleted versus control samples

  • For testis samples, CBY1-KO mice generated using approaches like the Cre-loxP system provide ideal negative controls

• Epitope blocking:

  • Pre-incubate antibody with recombinant CBY1 protein before staining

  • If the signal disappears, this confirms specific binding

  • Use recombinant human/mouse protein chibby homolog 1 protein (1-126AA) as blocking protein

• Multiple antibody approach:

  • Use antibodies targeting different CBY1 epitopes:

    • N-terminal regions (AA 1-41)

    • Middle regions (AA 41-97)

    • C-terminal regions (AA 97-126)

  • Concordant results with different antibodies strongly support specificity

• Signal correlation with expression level:

  • Compare staining intensity across tissues with known differential CBY1 expression

  • Verify that signal intensity correlates with expected expression patterns

• Demonstrating expected localization patterns:

  • Verify localization at ciliary bases in ciliated cells

  • Confirm co-localization with known CBY1 interacting partners like β-catenin or ciBAR1

• How does CBY1 localization change during ciliogenesis, and how can this be effectively tracked using antibodies?

CBY1 undergoes distinct localization changes during ciliogenesis that can be tracked using carefully designed immunofluorescence experiments:

• Early ciliogenesis stages:

  • CBY1 localizes to nascent centrioles

  • Co-localizes with Rabin8 (a guanine nucleotide exchange factor for Rab8)

  • Promotes recruitment of Rab8 for ciliary vesicle assembly

• Mature cilia:

  • Detected at the distal end of distal appendages in a ring-like pattern

  • Associates closely with ciliary membranes

  • Interacts with CEP164 at distal appendages

• Experimental tracking approach:

  • Use dual immunostaining with CBY1 antibody and centriole/basal body markers (e.g., γ-tubulin)

  • Include ciliary markers (acetylated tubulin, Arl13b) to track progression of ciliogenesis

  • Implement super-resolution microscopy techniques for precise localization

• Time-course experiments:

  • Synchronize ciliogenesis (serum starvation or cell cycle synchronization)

  • Fix cells at defined time points (0h, 6h, 12h, 24h, 48h)

  • Track CBY1 localization relative to centriole/basal body markers

• Recommended antibody combinations:

  • Anti-CBY1 (e.g., 12239-1-AP) with anti-acetylated tubulin and anti-γ-tubulin

  • For signal enhancement in weakly ciliated cells, use tyramide signal amplification

• What is the significance of CBY1 in ciliopathy research, and how can antibodies help elucidate disease mechanisms?

CBY1 plays crucial roles in ciliopathies, particularly Joubert syndrome, and antibodies can reveal several disease-relevant mechanisms:

• Loss-of-function (LOF) impact:

  • CBY1 LOF variants cause ciliopathies with features of Joubert syndrome

  • Patient fibroblasts show reduced ability to ciliate, increased ciliary length, and reduced levels of ciliary proteins AHI1 and ARL13B

  • Antibodies can detect these phenotypic changes in patient samples

• Experimental disease modeling:

  • CBY1 knockdown in zebrafish causes ciliopathy-related phenotypes

  • Antibodies can validate knockdown efficiency and track resulting ciliary defects

• Protein level assessment in patient samples:

  • CBY1 protein is undetectable by immunofluorescence in fibroblasts from patients with CBY1 mutations

  • This confirms transcript degradation through nonsense-mediated mRNA decay

• Complex formation analysis:

  • CBY1 forms complexes with ciBAR1 and ciBAR2

  • ciBAR1 is mutated in human ciliopathies including polydactyly

  • Co-immunoprecipitation with CBY1 antibodies can reveal altered complex formation in disease states

• Therapeutic target validation:

  • Rescue experiments with wild-type CBY1 versus mutant forms

  • Antibodies can confirm expression of rescue constructs and assess restoration of ciliary phenotypes

These findings highlight the importance of CBY1 antibodies in understanding ciliopathy disease mechanisms and potentially developing therapeutic approaches.

Research Application Scenarios

• How can CBY1 antibodies be used to investigate its role in cancer progression?

CBY1 antibodies can provide valuable insights into cancer biology through several experimental approaches:

• Expression level analysis in tumor samples:

  • Compare CBY1 expression between tumor and adjacent normal tissues using IHC

  • Correlate expression levels with clinical parameters (stage, grade, survival)

  • CBY1 functions as a Wnt/β-catenin antagonist, potentially acting as a tumor suppressor

• Subcellular localization studies:

  • Examine the distribution of CBY1 between nuclear and cytoplasmic compartments

  • CBY1, together with 14-3-3 proteins, facilitates cytoplasmic redistribution of β-catenin

  • Altered localization may indicate dysregulated Wnt signaling

• Functional pathway analysis:

  • CBY1 knockdown in SW480 colon cancer cells unexpectedly induces MET-like changes

  • These changes include increased E-cadherin and β-catenin at cell-cell contacts

  • Reduced anchorage-independent growth by 48% in soft agar assays

  • Chronic CBY1 knockdown may counteract tumor progression by promoting MET

• Cell migration assessment:

  • CBY1-knockdown cancer cells show epithelial-like migration patterns

  • They migrate as coherent epithelial sheets rather than individual cells

  • This altered migration pattern may impact metastatic potential

• Mechanistic interaction studies:

  • Use co-immunoprecipitation with CBY1 antibodies to identify cancer-relevant binding partners

  • Investigate how these interactions are altered in malignant versus non-malignant states

These approaches highlight the complex and context-dependent role of CBY1 in cancer, where both loss and gain of function may influence tumor behavior through different mechanisms.

• What are the best practices for quantifying CBY1 levels in tissue samples?

Accurate quantification of CBY1 in tissue samples requires careful methodological considerations:

• Sample preparation optimization:

  • For paraffin-embedded tissues, use citrate-based antigen retrieval (pH 9.0 preferred, pH 6.0 alternative)

  • For frozen sections, fix with 100% acetone for 10 minutes at -20°C

  • Standardize tissue thickness (5 μm for paraffin, 10 μm for frozen sections)

• Antibody selection and validation:

  • Use antibodies validated for the specific tissue and species being studied

  • Consider polyclonal antibodies (e.g., 12239-1-AP) for higher sensitivity

  • For human tissues, kidney and heart samples typically show reliable staining

• Internal controls and normalization:

  • Include positive control tissues (mouse liver/kidney) on the same slide

  • Use housekeeping proteins (β-actin, GAPDH) for Western blot normalization

  • For immunohistochemistry, compare to adjacent normal tissue within the same section

• Quantification methods:

  • For Western blot: Use densitometry with linear range validation

  • For IHC/IF: Implement digital image analysis with appropriate software

    • Measure staining intensity (integrated optical density)

    • Assess percent positive cells

    • Quantify subcellular distribution patterns

• Scoring systems for tissue microarrays:

  • Develop a standardized scoring system (0-3+) based on staining intensity

  • Consider both intensity and percentage of positive cells (H-score = Σ Pi × i, where i = intensity 0-3 and Pi = percentage of cells)

  • Use multiple independent scorers to ensure reliability

These practices ensure reproducible and meaningful quantification of CBY1 levels across diverse tissue samples and experimental conditions.