CIB1 Antibody

What is CIB1 and what are its primary cellular functions?

CIB1 is a 22 kDa calcium-binding protein that was initially identified as a binding partner of platelet integrin αIIb . This multifunctional protein serves as an endogenous inhibitor of agonist-induced integrin αIIbβ3 activation, playing a crucial role in regulating platelet function . Beyond integrin regulation, CIB1 physically associates with ASK1 (Apoptosis Signal-regulating Kinase 1) and inhibits its kinase activity, particularly during cellular responses to oxidative stress and TNF-α stimulation .

CIB1 contains EF-hand-like motifs in its N-terminal region that are involved in protein-protein interactions, as demonstrated through binding assays with ASK1 . Approximately one-fourth of total ASK1 exists in complex with CIB1 in cellular environments, highlighting the physiological relevance of this interaction . Furthermore, CIB1 has been shown to have neuroprotective functions, particularly against MPTP-induced neurotoxicity in Parkinson's disease models, through its inhibition of ASK1-mediated signaling pathways .

How should researchers select between different CIB1 antibodies for specific applications?

When selecting CIB1 antibodies, researchers should consider several critical factors:

Epitope recognition: Different antibodies may target distinct epitopes within the CIB1 protein. For example, some antibodies may recognize epitopes in the N-terminal region containing EF-hand-like motifs, which could potentially interfere with protein-protein interactions such as CIB1-ASK1 binding .

Application compatibility: Available commercial antibodies, such as the Mouse Anti-Human CIB1 Monoclonal Antibody (Clone #791119), have been validated for specific applications including Western blotting and immunohistochemistry . This particular antibody was developed against recombinant human CIB1 (Gly2-Leu191) and successfully detects CIB1 in human kidney tissue lysates and breast cancer tissue sections .

Species reactivity: Ensure the antibody recognizes CIB1 from your species of interest. The antibody described in the research data specifically targets human CIB1 , but requirements may differ depending on your model system.

Validation methods: Consider antibodies validated through multiple methods, including western blotting, immunoprecipitation, and testing in CIB1 knockout systems, as these provide greater confidence in specificity and performance .

What technical considerations are important when using CIB1 antibodies for protein interaction studies?

When studying CIB1 interactions with binding partners:

• Consider that some interactions may be calcium-dependent, as CIB1 is a calcium-binding protein .

• Be aware that standard lysis conditions may disrupt certain interactions. For example, CIB1-ASK1 interaction studies have been successfully performed using co-immunoprecipitation under conditions that preserve their association .

• Exposure to oxidative stress (H₂O₂) or TNF-α does not affect the extent of CIB1-ASK1 interaction, unlike other ASK1 binding partners such as thioredoxin . This stability should be considered when designing experiments.

• For detecting CIB1-integrin interactions, consider that CIB1 associates with only a portion of integrin αIIbβ3 molecules in the resting state, which may affect sensitivity requirements for detection methods .

What are the established protocols for Western blotting with CIB1 antibodies?

Based on validated research protocols, the following conditions are recommended for Western blot analysis of CIB1:

Sample preparation:

• Use PVDF membrane for protein transfer

• Perform experiments under reducing conditions

• Use appropriate lysis buffers that preserve CIB1 integrity (Immunoblot Buffer Group 1 has been validated)

Antibody conditions:

• Primary antibody concentration: 2 μg/mL of Mouse Anti-Human CIB1 Monoclonal Antibody

• Secondary antibody: HRP-conjugated Anti-Mouse IgG Secondary Antibody

• Expected band size: approximately 22 kDa

Critical controls:

• Positive control: Human kidney tissue lysate has been validated to express detectable levels of CIB1

• Negative control: Consider using RNAi-mediated CIB1 knockdown samples or tissues from CIB1 knockout models

The successful detection of CIB1 requires careful optimization of both blocking conditions and antibody dilutions to minimize background while maximizing specific signal intensity.

How can CIB1 antibodies be effectively used in immunohistochemistry studies?

For optimal immunohistochemical detection of CIB1 in tissue sections:

Tissue preparation:

• Use immersion-fixed, paraffin-embedded tissue sections

• Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic before antibody incubation

Staining protocol:

• Primary antibody concentration: 15 μg/mL of Mouse Anti-Human CIB1 Monoclonal Antibody

• Incubation conditions: Overnight at 4°C

• Detection system: HRP-DAB Cell & Tissue Staining Kit

• Counterstain: Hematoxylin

Localization pattern:

• In breast cancer tissue, specific CIB1 staining localizes to the cytoplasm of cancer cells

• This subcellular localization is consistent with CIB1's role as a cytoplasmic signaling protein

For multi-label immunofluorescence studies, researchers should carefully select compatible antibodies raised in different host species to avoid cross-reactivity during secondary antibody detection.

What methods are most effective for studying CIB1 interactions with binding partners?

Several complementary approaches have been successfully used to investigate CIB1 protein interactions:

Yeast two-hybrid screening:

• Effective for initial identification of CIB1 binding partners

• Has successfully identified interactions between CIB1 and ASK1

• Can determine interaction domains through truncation constructs (ASK1-NT and ASK1-ΔC bound to CIB1, while ASK1-ΔN did not)

In vitro binding assays:

• Direct binding demonstrated using GST-CIB1 fusion proteins and ³⁵S-labeled potential binding partners

• Identified that CIB1 binds directly to the NH₂-terminal regulatory domain of ASK1 (amino acids 378-648)

• Reciprocal binding assays revealed that ASK1 predominantly binds to the NH₂-terminal half of CIB1, which contains EF-hand-like motifs

Co-immunoprecipitation:

• Confirmed endogenous interaction between CIB1 and ASK1 in MCF7 cells

• Approximately one-fourth of total ASK1 existed in complex with CIB1

• Unlike the thioredoxin-ASK1 interaction, CIB1-ASK1 association was not affected by H₂O₂ or TNF-α treatment

Functional validation:

• In vitro kinase assays showed that GST-CIB1 inhibited ASK1 activity in immunoprecipitates from H₂O₂-treated cells

• Ectopic CIB1 expression decreased H₂O₂ or TNF-α-stimulated ASK1 activity

• RNAi-mediated CIB1 depletion potentiated ASK1, JNK1, and p38 MAPK activities in response to stress stimuli

How can researchers address specificity concerns with CIB1 antibodies?

To ensure antibody specificity and validate experimental results:

Genetic validation approaches:

• Use CIB1 knockout models as negative controls (CIB1⁻/⁻ mice or cell lines generated through serial gene targeting)

• Employ RNAi-mediated knockdown of CIB1 with appropriate controls

• Perform rescue experiments by expressing RNAi-resistant CIB1 constructs (as demonstrated with Flag₃-CIB1* in CIB1 siRNA-expressing cells)

Antibody validation techniques:

• Test antibody specificity in Western blots using recombinant CIB1 protein alongside cell/tissue lysates

• Perform peptide competition assays to confirm epitope specificity

• Compare results using multiple antibodies targeting different CIB1 epitopes

Control samples:

• When studying CIB1 in disease models, include appropriate controls (e.g., in MPTP-induced neurotoxicity studies, compare CIB1⁻/⁻ and wild-type mice)

• For cell culture experiments, include both positive controls (cells with confirmed CIB1 expression) and negative controls (CIB1-depleted cells)

How should researchers interpret conflicting data from CIB1 knockdown versus overexpression studies?

When faced with apparently contradictory results:

• Consider the biological context: CIB1 function may be cell-type and context-dependent. For example, CIB1 overexpression in DT40 cells did not significantly affect immunoglobulin gene conversion, suggesting that CIB1 levels are not limiting for this process in these cells .

• Evaluate expression levels: Determine whether CIB1 knockdown was complete or partial. In some studies, approximately 25% of ASK1 existed in complex with CIB1, suggesting that partial knockdown might not fully eliminate CIB1 function .

• Assess compensatory mechanisms: In CIB1 knockout models, compensatory upregulation of related proteins might mask phenotypes. CIB1-deficient DT40 cells showed no obvious growth defects, suggesting potential redundancy .

• Distinguish between acute versus chronic loss: RNAi-mediated acute depletion of CIB1 potentiated ASK1 activity in response to stress stimuli , while genetic knockout models might develop compensatory mechanisms during development.

• Validate key findings with multiple approaches: Combine overexpression, knockdown, and knockout approaches with interaction studies and functional assays to build a comprehensive understanding of CIB1 function.

What are the methodological considerations for studying CIB1 in neurodegenerative disease models?

When investigating CIB1's neuroprotective role:

Model selection:

• MPTP mouse models have successfully demonstrated CIB1's protective effect against dopaminergic neuron degeneration in Parkinson's disease

• Primary dopaminergic neurons treated with MPP⁺ provide a complementary in vitro model

Experimental design:

• Compare CIB1⁻/⁻ mice with wild-type controls following MPTP treatment

• Assess neurotoxicity through analysis of dopaminergic neuron survival and function

• For in vitro studies, use RNAi-mediated CIB1 depletion in primary neurons followed by MPP⁺ treatment

Mechanistic investigations:

• Examine CIB1's interaction with ASK1 in neuronal contexts

• Analyze activation of downstream signaling components (JNK and p38 MAPK pathways)

• Investigate whether neuroprotective interventions affect CIB1 expression or function

How might CIB1 antibodies be utilized in cancer research?

Recent findings suggest promising applications for CIB1 antibodies in cancer research:

Expression analysis:

• CIB1 has been detected in breast cancer tissues using immunohistochemistry, with specific staining localized to the cytoplasm of cancer cells

• Comparative analysis of CIB1 expression in normal versus malignant tissues could identify potential diagnostic or prognostic biomarkers

Functional studies:

• Given CIB1's role in regulating stress-response pathways via ASK1 inhibition , investigation of CIB1 function in cancer cell survival under stress conditions (e.g., chemotherapy, radiation) could reveal new therapeutic targets

• Correlation studies between CIB1 expression/localization and cancer progression might identify clinically relevant patterns

Therapeutic target assessment:

• CIB1 antibodies could help evaluate the effects of drugs targeting stress-response pathways in cancer cells

• Monitoring changes in CIB1-protein interactions following treatment could provide mechanistic insights into therapeutic responses

What are the considerations for developing phospho-specific CIB1 antibodies?

Although the search results do not specifically address phospho-specific CIB1 antibodies, developing such tools would require:

• Identification of physiologically relevant phosphorylation sites through mass spectrometry or prediction algorithms followed by site-directed mutagenesis

• Generation of phosphopeptide-specific antibodies using synthetic phosphopeptides corresponding to the identified sites

• Rigorous validation using phosphatase treatments and phospho-mimetic/phospho-dead mutants

• Correlation of phosphorylation status with functional outcomes such as protein-protein interactions or subcellular localization

Such antibodies could reveal how post-translational modifications regulate CIB1's interactions with binding partners like ASK1 or integrin αIIbβ3.

How can CIB1 antibodies contribute to understanding the interplay between calcium signaling and stress responses?

As a calcium-binding protein that regulates stress-response pathways, CIB1 represents an important link between calcium signaling and cellular stress responses:

Calcium-dependent interactions:

• CIB1 antibodies could be used to investigate whether calcium levels affect CIB1's interactions with binding partners like ASK1

• Co-immunoprecipitation studies under varying calcium concentrations might reveal calcium-sensitive protein complexes

Subcellular localization changes:

• Immunofluorescence with CIB1 antibodies could track potential calcium-induced changes in CIB1 localization during stress responses

• Multi-label imaging with markers of calcium signaling organelles would provide spatial context

Stress-response pathway integration:

• CIB1 inhibits ASK1-mediated stress signaling , suggesting that calcium signals might modulate stress responses through CIB1

• Simultaneous monitoring of calcium levels, CIB1 status, and downstream stress kinases could reveal integrated signaling mechanisms

By applying these approaches, researchers can gain deeper insights into how CIB1 functions at the intersection of calcium homeostasis and cellular stress adaptation pathways.