spred2 Antibody

What is SPRED2 and what are its major functions in signaling pathways?

SPRED2 (Sprouty-related, EVH1 domain-containing protein 2) is a member of the Sprouty family that acts as a negative regulator during development. The protein primarily functions by suppressing the phosphorylation and activation of RAF, thereby inhibiting the ERK signaling pathway . SPRED2 plays a critical role in the negative feedback regulation of FGF signaling during embryogenesis and angiogenesis . Additionally, SPRED2 has been implicated in tumor suppression, as its expression levels are inversely correlated with tumor invasion and metastasis in human hepatocellular carcinoma, suggesting potential utility as a prognostic factor .

What are the essential structural domains of SPRED2 and their functional significance?

SPRED2 contains several critical domains that mediate its regulatory functions:

• N-terminal EVH1 domain: Essential for SPRED2's inhibitory activity on ERK1/2 signaling and mediates protein-protein interactions with partners like NBR1

• SPRY domain: Works in conjunction with the EVH1 domain for full functionality in ERK inhibition

• KBD (Kinase Binding Domain): Present in SPRED2 but dispensable for interaction with NBR1 and for ERK1/2 inhibition

Studies using mutant SPRED2 constructs have demonstrated that both EVH1 and SPRY domains are required for effective interaction with NBR1 and subsequent inhibition of ERK1/2 activity, while the KBD appears to be nonessential for these specific functions .

How does SPRED2 specifically regulate the ERK/MAPK signaling pathway?

SPRED2 regulates ERK/MAPK signaling through a mechanism involving interaction with the late endosomal protein NBR1. This interaction is mediated by SPRED2's EVH1 domain and is critical for its inhibitory function . The functional process involves:

• SPRED2 binds NBR1 through its EVH1 domain

• This complex formation downregulates FGF2-mediated ERK1/2 activity at various time points after stimulation

• Co-expression of NBR1 with SPRED2 enhances SPRED2-mediated ERK1/2 inhibition

• siRNA-mediated depletion of endogenous NBR1 significantly reduces SPRED2's inhibitory effect on ERK1/2

These findings indicate that SPRED2 requires NBR1 to effectively inhibit ERK1/2 signaling, highlighting a previously unrecognized mechanism of ERK pathway regulation.

What criteria should researchers consider when selecting a SPRED2 antibody for specific applications?

When selecting a SPRED2 antibody, researchers should evaluate:

• Application suitability: Confirm the antibody has been validated for your specific application (Western blot, immunoprecipitation, etc.)

• Specificity: Choose antibodies specifically validated for SPRED2 with minimal cross-reactivity to related proteins like SPRED1 or SPRED3

• Immunogen information: Consider the epitope location - antibodies raised against different regions may have different detection capabilities

• Host species: Select appropriate host species to avoid cross-reactivity issues in your experimental system

• Clonality: Polyclonal antibodies may offer broader epitope recognition, while monoclonal antibodies provide greater specificity

• Validation data: Review available validation data showing specific detection in relevant tissues or cell types

For example, commercially available SPRED2 antibodies raised against peptides near the center of human SPRED2 have been successfully used in Western blotting applications and show minimal cross-reactivity with other SPRED family members .

How can researchers validate the specificity of SPRED2 antibodies?

A comprehensive validation approach for SPRED2 antibodies should include:

• Western blot analysis comparing SPRED2-expressing and SPRED2-depleted samples (siRNA knockdown or knockout cells)

• Peptide competition assays to confirm epitope specificity

• Testing in multiple cell types/tissues with known SPRED2 expression patterns

• Comparing detection patterns with multiple SPRED2 antibodies targeting different epitopes

• Recombinant protein expression controls using wild-type and mutant SPRED2 constructs

Evidence from published research demonstrates that validated SPRED2 antibodies can detect specific bands at the expected molecular weight in human small intestine tissue lysates, with signal intensity proportional to antibody concentration (1 μg/mL versus 2 μg/mL) .

What controls are essential for experiments using SPRED2 antibodies?

For rigorous SPRED2 antibody experiments, implement these controls:

• Positive controls: Include lysates from tissues or cell lines with confirmed SPRED2 expression (e.g., human small intestine tissue)

• Negative controls: Use SPRED2 knockout/knockdown samples or tissues known not to express SPRED2

• Loading controls: Employ housekeeping proteins (β-actin, GAPDH) to normalize protein loading

• Antibody controls: Include secondary-only controls to identify non-specific binding

• Isotype controls: Use matched isotype antibodies to identify Fc receptor-mediated binding

• Peptide competition: Pre-incubation with immunizing peptide should abolish specific binding

• Expression controls: When studying overexpressed SPRED2, include empty vector controls

When testing SPRED2 variants or mutants, wild-type SPRED2 expression should be included as a functional reference control .

What is the optimal protocol for SPRED2 detection by Western blotting?

For optimal Western blot detection of SPRED2:

• Sample preparation:

  • Lyse cells in RIPA buffer (pH 8.0) containing 20 mM NaF, 1 mM Na₃VO₄, and protease inhibitors

  • Incubate lysates on ice for 30 minutes

  • Centrifuge at 16,000 × g for 20 minutes at 4°C

  • Collect supernatants and determine protein concentration using bicinchoninic acid assay

• SDS-PAGE and transfer:

  • Resolve proteins on 10% SDS-polyacrylamide gel

  • Transfer to nitrocellulose membrane using the Trans-Blot Turbo transfer system

• Immunoblotting:

  • Block membrane with 5% non-fat milk powder in PBS containing 0.1% Tween-20 for 1 hour

  • For SPRED2 level analyses, incubate with primary antibody for 1 hour

  • For phosphorylation studies (pRAF1/MEK/ERK) or co-immunoprecipitation assays, incubate overnight

  • Use secondary antibodies diluted in blocking solution

  • Detect immunoreactive proteins using enhanced chemiluminescence (ECL)

For quantification, perform densitometric analysis of protein bands using appropriate image analysis software .

How should researchers optimize co-immunoprecipitation assays to study SPRED2 protein interactions?

For effective co-immunoprecipitation of SPRED2 and its binding partners:

• Cell preparation:

  • Transfect HEK293T cells with wild-type or variant SPRED2 constructs

  • Serum-starve cells prior to stimulation

  • Stimulate with appropriate ligand (e.g., EGF at 30 ng/mL)

• Lysis and pre-clearing:

  • Lyse cells in IP buffer containing 25 mM Tris-HCl (pH 7.4), 1% Triton X-100, 2 mM EDTA (pH 8.0), 150 mM NaCl, and protease inhibitors

  • Centrifuge at 10,000 × g for 20 minutes at 4°C

  • Collect supernatants and determine protein concentration

• Immunoprecipitation:

  • Use equal amounts of total proteins for immunoprecipitation

  • Immunoprecipitate using anti-tag antibody (e.g., anti-Xpress) cross-linked to Protein G Sepharose beads

  • Incubate for 2 hours at 4°C

  • Recover beads by centrifugation

  • Wash six times with IP buffer

• Elution and analysis:

  • Elute immunoprecipitated proteins with sample buffer by incubating at 95°C for 5 minutes

  • Adjust loading volumes based on expression levels (e.g., WT SPRED2: 10 μL; variant SPRED2: 30 μL)

  • Normalize immunoprecipitated endogenous partners to the amount of immunoprecipitated SPRED2 proteins

This protocol has been successfully used to demonstrate SPRED2 interaction with endogenous neurofibromin.

What methods are recommended for analyzing SPRED2's role in regulating ERK signaling?

To effectively analyze SPRED2's impact on ERK signaling:

• Experimental design:

  • Compare ERK1/2 phosphorylation at various time points after growth factor stimulation (e.g., FGF2)

  • Test multiple SPRED2 constructs (wild-type and domain mutants) to identify critical regions

  • Co-express potential binding partners (e.g., NBR1) to assess cooperative effects

• ERK activity assays:

  • Express myc-tagged SPRED2 in appropriate cell lines (e.g., 293T cells)

  • Stimulate with growth factors and collect samples at defined time points

  • Analyze phospho-ERK1/2 levels by immunoblotting

  • Include domain mutants (EVH1, SPRY, KBD) to determine domain-specific contributions to inhibition

• Binding partner cooperation:

  • Express NBR1 alone or co-express with SPRED2

  • Assess ERK1/2 activity under these conditions

  • Compare to SPRED2 expression alone to identify enhanced inhibition

• Knockdown studies:

  • Use siRNA to deplete endogenous binding partners (e.g., NBR1)

  • Express SPRED2 using inducible constructs after sufficient depletion

  • Measure ERK1/2 activity to determine the dependence of SPRED2 function on binding partners

Published data demonstrates that while NBR1 alone does not reduce ERK1/2 activity, it enhances SPRED2-mediated inhibition when co-expressed, and knockdown of NBR1 significantly reduces SPRED2's inhibitory capacity .

How can researchers investigate SPRED2's role in epithelial-mesenchymal transition (EMT)?

To study SPRED2's involvement in EMT regulation:

• Experimental approach:

  • Overexpress SPRED2 in appropriate cancer cell lines (e.g., SW480 and HCT116 for colorectal cancer)

  • Use adenoviral vectors (Ad.Spred2) with empty vector controls (Ad.Null)

  • Collect cells at multiple time points (e.g., 24 and 48 hours post-infection)

• Protein analysis:

  • Extract proteins using direct lysis buffer

  • Analyze expression of:

    • SPRED2 (to confirm overexpression)

    • ERK and phosphorylated ERK (p-ERK) to assess pathway inhibition

    • EMT markers: E-cadherin, N-cadherin, vimentin

    • TGF-β pathway components: SMAD2/3, phosphorylated SMAD2/3 (p-SMAD2/3), SMAD4

• Functional assays:

  • Migration and invasion assays to assess EMT-associated phenotypes

  • Cell morphology assessment

  • Expression analysis of EMT-related transcription factors

Research has demonstrated that SPRED2 inhibits EMT in colorectal cancer cells primarily by blocking the ERK signaling pathway, sometimes with concurrent reduction in TGFβ/SMAD signaling .

What approaches can be used to investigate SPRED2 subcellular localization and its functional significance?

For studying SPRED2 subcellular localization:

• Experimental design:

  • Express wild-type and mutant SPRED2 constructs in relevant cell lines

  • Apply appropriate stimulation (e.g., growth factor treatment after starvation)

  • Use confocal microscopy to track protein localization

• Co-localization studies:

  • Stain for endosomal markers (e.g., mannose 6-phosphate receptor for late endosomes)

  • Investigate co-localization with binding partners (e.g., neurofibromin GRD domain)

  • Analyze effects of domain mutations on localization patterns

• Live cell imaging:

  • Create fluorescently tagged SPRED2 constructs

  • Track dynamic localization changes following stimulation

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to assess protein mobility

• Biochemical fractionation:

  • Separate cellular compartments (cytosol, membrane, nuclear fractions)

  • Analyze SPRED2 distribution across fractions

  • Assess how mutations or stimulation alter this distribution

Previous studies have shown that SPRED2 co-localizes with late endosomal markers, and this localization pattern is dependent on its EVH1 and SPRY domains, which are also essential for interaction with binding partners like NBR1 .

How does SPRED2 loss-of-function contribute to Noonan syndrome-like phenotypes?

To investigate SPRED2's role in Noonan syndrome-like conditions:

• Genetic analysis approaches:

  • Sequence SPRED2 in patients with Noonan syndrome-like features lacking mutations in known causative genes

  • Focus on consanguineous families with suspected recessive inheritance patterns

  • Use extended gene panels designed for RASopathy testing

• Functional characterization of patient variants:

  • Create expression constructs containing patient-specific SPRED2 variants

  • Assess protein stability and degradation rates

  • Evaluate binding to known interaction partners (e.g., neurofibromin)

  • Measure impact on ERK pathway regulation

• Cellular phenotype analysis:

  • Compare signaling profiles between cells expressing wild-type vs. variant SPRED2

  • Assess impact on cell morphology, proliferation, and differentiation

  • Evaluate response to growth factor stimulation

Research has identified bi-allelic variants in SPRED2 causing a disorder resembling Noonan syndrome, expanding our understanding of the genetic basis of RASopathies and highlighting SPRED2's critical role in developmental signaling regulation .

What are common challenges in detecting SPRED2 and how can they be addressed?

Common challenges and solutions in SPRED2 detection:

• Low endogenous expression:

  • Use tissues with confirmed high expression (e.g., human small intestine)

  • Concentrate proteins through immunoprecipitation before detection

  • Employ more sensitive detection methods (e.g., chemiluminescent substrates with longer exposure times)

• Antibody specificity issues:

  • Select antibodies specifically validated for SPRED2 with minimal cross-reactivity to SPRED1/SPRED3

  • Include appropriate positive and negative controls

  • Consider using multiple antibodies targeting different epitopes

• Protein degradation:

  • Include protease inhibitors in all buffers

  • Work at 4°C when possible

  • For unstable SPRED2 variants, adjust loading volumes (e.g., wild-type: 10 μL; variant: 30 μL)

• Background issues:

  • Optimize blocking conditions (test BSA vs. non-fat milk)

  • Increase washing steps and duration

  • Titrate primary antibody concentration (test range: 1-2 μg/mL)

How can researchers effectively study SPRED2 interactions with binding partners like NBR1?

To optimize studies of SPRED2-partner interactions:

• Binding domain mapping:

  • Create domain deletion/mutation constructs (e.g., EVH1, SPRY, KBD deletions)

  • Use yeast two-hybrid screening with the EVH1 domain as bait to identify novel partners

  • Validate interactions through multiple methods (co-IP, proximity ligation, FRET)

• Functional validation:

  • Co-express SPRED2 with binding partners to test cooperative effects

  • Use siRNA to deplete endogenous partners and assess impact on SPRED2 function

  • Create domain-specific mutations that disrupt specific interactions

• Visualization techniques:

  • Perform co-localization studies using confocal microscopy

  • Use fluorescently tagged constructs to visualize interaction dynamics

  • Apply super-resolution microscopy for detailed localization analysis

Research has established that NBR1 interacts with SPRED2's EVH1 domain, enhancing SPRED2-mediated ERK1/2 inhibition when co-expressed, while NBR1 depletion significantly reduces SPRED2's inhibitory function .

What considerations are important when studying SPRED2's role in cancer progression and metastasis?

For investigating SPRED2 in cancer contexts:

• Expression analysis:

  • Compare SPRED2 levels between tumor and matched normal tissues

  • Correlate expression with clinical parameters (staging, invasion, metastasis)

  • Consider analysis across cancer subtypes and progression stages

• Mechanism studies:

  • Investigate SPRED2's impact on cancer-related signaling pathways:

    • ERK/MAPK pathway inhibition

    • TGFβ/SMAD signaling regulation

    • EMT marker modulation (E-cadherin, N-cadherin, vimentin)

• Functional assessments:

  • Manipulate SPRED2 expression in cancer cell lines and assess:

    • Proliferation and colony formation

    • Migration and invasion capabilities

    • Anchorage-independent growth

    • In vivo tumor formation and metastasis

• Clinical correlation:

  • Analyze patient samples for SPRED2 expression/mutation

  • Correlate findings with patient outcomes

  • Consider SPRED2 as a potential prognostic biomarker