SH2B1 Antibody

What is SH2B1 and what cellular processes does it regulate?

SH2B1 is an adaptor protein that plays crucial roles in multiple signaling pathways, particularly in cytokine and growth factor signaling. Research demonstrates that SH2B1 associates with the erythropoietin receptor (EPO-R) and functions as a negative regulator of EPO-mediated signal transduction . Additionally, SH2B1 is highly expressed in pancreatic β-cells where it regulates glucose metabolism by promoting β-cell survival and islet expansion . It enhances insulin and IGF-1 signaling through the PI 3-kinase/Akt pathway, functioning as a prosurvival protein that protects β-cells against injury and apoptosis . Mice express four isoforms of SH2B1 (α, β, γ, and δ) via alternative mRNA splicing, with multiple forms detectable in pancreatic tissue .

How should I select the appropriate SH2B1 antibody for my experimental application?

Selection of an appropriate SH2B1 antibody depends on your specific experimental application and the isoform(s) you wish to detect. When selecting an antibody, consider the following methodological approach:

• Determine target specificity requirements:

  • For detection of all isoforms, select antibodies targeting shared regions (amino acids 1-436 encode all four SH2B1 isoforms)

  • For isoform-specific detection, choose antibodies raised against unique C-terminal regions

• Application compatibility:

  • For immunoprecipitation experiments, purified polyclonal antibodies have been successfully used

  • For immunoblotting, antibodies that specifically recognize epitopes corresponding to amino acids 527-670 of SH2B1β have proven effective

• Validation status:

  • Confirm the antibody has been validated in your experimental system

  • Use appropriate controls (SH2B1 knockout tissues show no staining with specific antibodies)

What are the recommended protocols for SH2B1 immunoprecipitation?

Based on published methodologies, the following protocol is recommended for immunoprecipitation of SH2B1:

• Cell preparation:

  • Deplete cells of cytokine for 4 hours prior to stimulation

  • Stimulate with appropriate factor (e.g., EPO or vehicle control)

• Immunoprecipitation procedure:

  • Use purified anti-SH2B1 antibodies that recognize amino acids 527-670 of SH2B1β

  • Perform immunoprecipitation followed by Western blotting with anti-phosphotyrosine antibody (e.g., 4G10)

  • When investigating SH2B1 interaction with EPO-R, confirm using Tyr(P)-343-EPO-R specific antibody for immunoblotting

• Controls:

  • Include unstimulated cells as negative control

  • Use cells stimulated with alternative cytokines (e.g., IL-3) for specificity control

This approach has successfully demonstrated that SH2B1 co-immunoprecipitates with phosphorylated EPO-R in multiple cell types, including Ba/F3, DA-3, HCD-57 cells, and primary splenic erythroblasts .

How can SH2B1 antibodies be utilized to investigate signaling pathway interactions?

SH2B1 antibodies can be strategically employed to dissect complex signaling networks through the following methodological approaches:

• Signaling cascade analysis:

  • Use SH2B1 antibodies in immunoprecipitation followed by immunoblotting with phospho-specific antibodies targeting key signaling proteins

  • Research has revealed that SH2B1 affects both PI 3-kinase/Akt and ERK1/2 pathways in response to IGF-1 and insulin stimulation

• Temporal signaling dynamics:

  • Perform time-course experiments with SH2B1 immunoprecipitation at multiple time points after stimulation

  • Analyze changes in associated proteins to map temporal signaling patterns

• Domain-specific interactions:

  • Utilize GST-SH2B1 fusion proteins containing specific domains (e.g., SH2 domain) to identify domain-dependent interactions

  • Research has demonstrated that the SH2 domain is required for SH2B1 association with activated EPO-R in both cultured cells and primary erythroblasts

What are the considerations for using SH2B1 antibodies in tissue-specific knockout models?

When using SH2B1 antibodies to validate and characterize tissue-specific knockout models, researchers should implement the following methodological approach:

• Validation of knockout efficiency:

  • Perform immunoblotting of target tissues using antibodies that recognize regions deleted in the knockout construct

  • In pancreas-specific SH2B1 knockout (PKO) mice, antibodies confirmed absence of SH2B1 protein in pancreatic tissue while preserving expression in other tissues like brain and liver

• Cross-tissue expression analysis:

  • Use immunohistochemistry with SH2B1 antibodies to map expression patterns within target and control tissues

  • Research has shown that SH2B1 is predominantly colocalized with insulin in β-cells, with minimal expression in acinar tissue throughout the pancreas

• Functional validation:

  • Combine antibody-based protein detection with functional assays

  • For example, in PKO mice, decreased SH2B1 expression correlates with impaired activation of the PI 3-kinase/Akt pathway, increased β-cell apoptosis, and exacerbated glucose intolerance

• Control selection:

  • Include appropriate genetic controls (e.g., SH2B1 f/f mice without Cre expression)

  • Use global knockout tissues as negative controls for antibody specificity

How can I design experiments to study the role of SH2B1 in signal transduction using antibody-based approaches?

When designing experiments to investigate SH2B1's role in signal transduction, implement the following methodological framework:

• Genetic manipulation strategies:

  • For knockdown studies, use validated shRNA sequences (e.g., 5′-CATCTGTGGTTCCAGTCCA-3′) that effectively reduce SH2B1 expression by ~60%

  • For overexpression studies, use stable cell lines expressing SH2B1β

• Signaling pathway assessment:

  • Stimulate cells with relevant factors (e.g., IGF-1, insulin, EPO) following appropriate serum starvation

  • Analyze phosphorylation of downstream effectors (Akt at Thr308 and Ser473, ERK1/2) using phospho-specific antibodies

  • Compare phosphorylation patterns between SH2B1-manipulated cells and appropriate controls

• Functional outcome measurements:

  • Assess cell viability using colorimetric assays like MTT following treatment with stressors (e.g., STZ)

  • Quantify apoptosis using TUNEL assays

  • Integrate signaling data with functional outcomes to establish causality

What controls should be included when using SH2B1 antibodies in immunohistochemistry?

When performing immunohistochemistry with SH2B1 antibodies, incorporate the following controls to ensure reliable and interpretable results:

• Genetic negative controls:

  • Include tissue sections from SH2B1 knockout models where the protein is absent

  • Research has confirmed that no SH2B1 staining is detected in islets from SH2B1 KO mice, providing definitive validation of antibody specificity

• Co-localization controls:

  • Perform dual immunostaining with markers of specific cell types

  • Studies have demonstrated SH2B1 predominantly colocalizes with insulin in β-cells, with some expression in insulin-negative cells in the islet mantle

• Technical controls:

  • Include secondary antibody-only controls to assess non-specific binding

  • Use isotype-matched irrelevant primary antibodies to evaluate background staining

• Tissue type controls:

  • Include multiple tissue types with known differential expression

  • Research has shown minimal SH2B1 staining in acinar tissue compared to islets, providing an internal negative control

How should I interpret contradictory data when using different SH2B1 antibodies?

When confronted with contradictory results from different SH2B1 antibodies, implement this systematic approach:

• Epitope mapping analysis:

  • Compare the epitopes recognized by each antibody

  • Antibodies targeting different regions (e.g., N-terminal vs. C-terminal) may detect different isoforms or phosphorylation states

• Validation hierarchy:

  • Prioritize data from antibodies validated with knockout controls

  • SH2B1 knockout tissues should show complete absence of signal with specific antibodies

• Isoform-specific considerations:

  • Determine if contradictions arise from differential isoform detection

  • Mice express four SH2B1 isoforms (α, β, γ, and δ) through alternative splicing

  • Use isoform-specific positive controls when available

• Resolution strategies:

  • Employ multiple detection methods (e.g., immunoblotting, immunoprecipitation, immunohistochemistry)

  • Use functional approaches (e.g., siRNA knockdown) to complement antibody-based findings

  • Consider epitope-masking effects from protein-protein interactions

What are the critical variables that affect SH2B1 antibody performance in immunoprecipitation experiments?

Based on published methodologies, the following variables significantly impact SH2B1 antibody performance in immunoprecipitation experiments:

• Cell stimulation conditions:

  • Cytokine depletion duration (optimal: 4 hours for primary cells)

  • Stimulation agent and concentration (EPO, IGF-1, or insulin)

  • Stimulation timing (transient vs. sustained signaling)

• Lysis conditions:

  • Buffer composition and detergent selection

  • Phosphatase inhibitor inclusion is critical for preserving phosphorylation states

  • Temperature and duration of extraction

• Antibody characteristics:

  • Affinity purification improves specificity (protein-A-Sepharose columns have been used successfully)

  • Antibody concentration and incubation conditions

  • Polyclonal antibodies targeting amino acids 527-670 of SH2B1β have proven effective for immunoprecipitation

• Detection strategy:

  • Using phospho-specific antibodies (e.g., 4G10 for phosphotyrosine) for immunoblotting

  • Using target-specific antibodies (e.g., anti-Tyr(P)-343-EPO-R) to confirm co-immunoprecipitated proteins

How can SH2B1 antibodies be utilized to investigate β-cell dysfunction in metabolic disorders?

SH2B1 antibodies can be strategically employed to explore β-cell dysfunction through these methodological approaches:

• Tissue-specific expression analysis:

  • Use immunohistochemistry with SH2B1 antibodies to analyze expression patterns in pancreatic sections from normal vs. diseased states

  • Research has established that SH2B1 is highly expressed in islets compared to acinar tissue

• Signaling pathway assessment:

  • Use phospho-specific antibodies to evaluate SH2B1's impact on insulin/IGF-1 signaling

  • Data show SH2B1 enhances the PI 3-kinase/Akt pathway, which is critical for β-cell survival

  • Compare signaling in normal vs. diabetic models to identify alterations

• Stress response characterization:

  • Analyze SH2B1's role in protecting against β-cell stressors relevant to diabetes

  • Research has shown that SH2B1 protects against STZ-induced apoptosis

  • Use TUNEL assays combined with SH2B1 detection to correlate expression with survival

• Genetic model integration:

  • Utilize pancreas-specific SH2B1 knockout models to study functional outcomes

  • PKO mice demonstrate increased β-cell apoptosis, decreased β-cell proliferation, decreased insulin content, and exacerbated glucose intolerance when challenged with high-fat diet

What methods can be employed to study SH2B1 isoform-specific functions using antibody-based approaches?

To investigate isoform-specific functions of SH2B1, implement these methodological strategies:

• Isoform-selective antibody development:

  • Generate antibodies targeting unique C-terminal regions that differentiate between SH2B1 isoforms (α, β, γ, and δ)

  • Validate specificity using overexpression systems with individual isoforms

• Expression profiling:

  • Use isoform-specific antibodies to map differential expression across tissues and cell types

  • Research has detected multiple SH2B1 forms in pancreatic extracts, with relatively higher levels in islets than in total pancreatic extracts

• Functional rescue experiments:

  • In SH2B1-silenced cells or knockout models, reintroduce specific isoforms

  • For example, SH2B1β overexpression in INS-1 832/13 cells enhances cell viability and reduces apoptosis

  • Use antibodies to confirm expression and correlate with functional outcomes

• Domain interaction mapping:

  • Use GST-SH2B1 fusion proteins containing specific domains

  • In vitro mixing experiments have demonstrated that the SH2 domain is required for SH2B1 association with phosphorylated EPO-R

  • Compare binding partners between different isoforms using co-immunoprecipitation followed by mass spectrometry