FBP2 Antibody

What is FBP2 and what are its primary cellular functions?

FBP2, also called muscle FBP, is a multifunctional enzyme that catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate in the presence of divalent cations . Beyond its metabolic role in gluconeogenesis, FBP2 demonstrates remarkable compartmentalization with distinct functions in different cellular locations:

• In the cytoplasm: Functions primarily as a gluconeogenic enzyme participating in glycogen synthesis from carbohydrate precursors

• In mitochondria: Protects against stress-induced depolarization and regulates mitochondrial motility and shape

• In the nucleus: Regulates gene expression related to mitochondrial biogenesis and oxidative phosphorylation

This multifunctionality makes FBP2 an important research target across fields including metabolism, cancer biology, and cellular physiology.

What is known about the oligomeric states of FBP2 and their significance?

Research has revealed that FBP2 exists in an equilibrium between tetrameric and dimeric forms, challenging the traditional view that FBP2 is strictly tetrameric . The dimeric form is fully active and insensitive to AMP, while the tetrameric form can be inhibited by AMP. At physiological AMP concentrations (0.16 μM), wild-type FBP2 is predominantly tetrameric .

The oligomeric state appears functionally significant, as:

• The dimeric form of FBP2 binds to mitochondria and has protective functions

• Residues D187 and L190 are crucial for forming interactions between dimers (the "leucine lock") which stabilizes the active tetrameric form of the enzyme (R-state)

• Mutations affecting these residues (D187L and L190G) significantly impact AMP inhibition and tetramerization

Understanding these oligomeric states is crucial when designing experiments to study FBP2 function and regulation.

How does FBP2 impact mitochondrial function?

FBP2 plays a significant role in mitochondrial regulation:

• Promotes cell survival under oxidative stress conditions

• Protects mitochondria from stress-induced depolarization

• Influences mitochondrial motility and shape

• Regulates mitochondrial biogenesis in certain contexts

Studies show cellular ROS production is inversely proportional to FBP2 expression levels, while mitochondrial polarization is directly proportional to FBP2 expression . Interestingly, in sarcoma cells, FBP2 restoration reduces mitochondrial DNA content, mitochondrial mass, and citrate synthase activity, indicating complex context-dependent effects on mitochondrial function .

What are the optimal conditions for immunofluorescence studies using FBP2 antibodies?

For successful immunofluorescence detection of FBP2:

• Fix cells in 4% formaldehyde

• Permeabilize using 0.2% Triton X-100

• Block in 10% normal goat serum

• Incubate with primary FBP2 antibody (e.g., rabbit polyclonal antibodies have been validated at dilutions around 1/166)

• Use appropriate fluorophore-conjugated secondary antibodies (Alexa 633 or FITC-conjugated)

• Mount cells in Fluoroshield with DAPI for nuclear counterstaining

• For mitochondrial co-localization studies, include appropriate mitochondrial markers (e.g., anti-TOMM antibody)

For quantitative analysis of FBP2 co-localization with cellular structures like mitochondria or microtubules, the Manders' coefficient (M) can be determined using the JACoP plugin of ImageJ/FIJI . This coefficient ranges from 0 (no co-localization) to 1 (100% co-localization).

How can I investigate FBP2's interactions with other proteins?

Proximity Ligation Assay (PLA) has proven effective for studying FBP2 protein-protein interactions:

• Use DuoLink® In Situ Orange Starter Kit (Mouse/Rabbit)

• Apply appropriate primary antibodies (e.g., anti-phospho-Tau, anti-Tau, anti-MAP1B with anti-FBP2 antibodies)

• Always include control reactions where primary antibodies are omitted

• Mount cells in Fluoroshield with DAPI

• Perform experiments in triplicate with measurements from at least 300 cells per condition

This technique allows visualization and quantification of interactions between FBP2 and other proteins with high specificity and spatial resolution.

What controls should I include when studying FBP2 in different cellular compartments?

When examining compartment-specific FBP2:

• Include appropriate subcellular markers:

  • Mitochondria: TOMM20, cytochrome c

  • Nucleus: DAPI staining, nuclear fraction markers

  • Cytosol: Cytosolic markers to confirm fractionation quality

• For genetic manipulation studies, include:

  • Wild-type cells for baseline comparison

  • FBP2-silenced cells (FBP2-) as negative controls

  • FBP2-overexpressing cells (FBP2+) for gain-of-function studies

• Consider using catalytically inactive FBP2 mutants (e.g., G260R) to distinguish between enzymatic and non-enzymatic functions of FBP2

• For nuclear localization studies, ensure nuclear signal is properly distinguished from cytoplasmic signal

How can I study the different oligomeric states of FBP2?

To investigate FBP2 oligomeric states:

• Use native PAGE rather than denaturing SDS-PAGE to preserve oligomeric structure

• Preincubate samples with varying AMP concentrations (0-5 mM) to observe effects on oligomerization

• Consider using the L190G mutation as an experimental tool, as it maintains FBP2 in a dimeric state regardless of AMP concentration

• The D187L mutation provides an intermediate phenotype with partial resistance to AMP-induced tetramerization

• When designing antibody-based detection, ensure the epitope is accessible in both oligomeric states

For functional studies, note that different FBP2 mutants require different Mg²⁺ concentrations for optimal activity: 5 mM for wild-type FBP2, 15 mM for D187L mutant, and 10 mM for L190G mutant .

How can I use FBP2 antibodies to investigate its role in cancer biology?

For studying FBP2's tumor suppressive functions:

• Use doxycycline-inducible systems for controlled FBP2 expression in cancer cell lines

• Compare expression levels to appropriate control cells (e.g., HSMM for liposarcoma studies)

• Assess effects on:

  • Cell proliferation under various nutrient conditions (low serum, low glucose)

  • Anchorage-independent growth in 3D soft agar colony assays

  • Tumor growth in mouse xenograft models

• Measure tumor cell proliferation markers (e.g., phospho-histone H3)

• Analyze mitochondrial parameters:

  • Mitochondrial DNA content by qPCR (mitochondrial to nuclear DNA ratio)

  • Mitochondrial mass using flow cytometry with MitoTracker

  • Citrate synthase activity

  • Ultrastructural changes using transmission electron microscopy

Research has demonstrated that FBP2 restoration inhibits sarcoma growth through dual mechanisms: inhibiting glycolysis associated with the Warburg effect and restraining mitochondrial biogenesis and respiration .

What techniques can I use to investigate FBP2's nuclear functions?

To study FBP2's nuclear activities:

• Perform subcellular fractionation to isolate nuclear and cytosolic fractions

• Verify FBP2 nuclear localization using immunofluorescence with DAPI co-staining

• For functional analysis, conduct RNA-seq on cells with and without FBP2 expression

• Use computational analysis tools like Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to identify affected pathways

• Focus on pathways related to OXPHOS, mitochondrial function, and cell cycle

• Validate findings with qRT-PCR for key genes identified in the RNA-seq

Studies have shown that nuclear FBP2 influences expression of genes involved in oxidative phosphorylation and mitochondrial function, with RNA-seq revealing reduced gene expression signatures for E2F targets, MYC targets, G2M checkpoint, and OXPHOS in FBP2-restored cells .

How can I optimize experiments to study FBP2's differential effects under varying stress conditions?

To investigate FBP2's context-dependent functions:

• Compare cellular responses under:

  • Normal conditions

  • Oxidative stress (H₂O₂ treatment)

  • Hypoxic conditions

  • Reoxygenation after hypoxia

• Measure multiple parameters:

  • Cell viability (MTT assay)

  • Mitochondrial membrane potential

  • ROS production

  • Cell proliferation markers

• Compare cells with different FBP2 expression levels and mutants:

  • Wild-type FBP2

  • FBP2-silenced cells

  • FBP2-overexpressing cells

  • Cells expressing D187L and L190G mutants

Research has shown that while FBP2 promotes cell survival under oxidative stress, it impairs survival under hypoxic conditions - an important consideration for tumor microenvironments . Under H₂O₂ treatment, FBP2+ cells were approximately 1.9 times more viable than wild-type cells and 3.7 times more viable than FBP2- cells .

Why might I observe inconsistent results with FBP2 antibodies in different experimental contexts?

Inconsistent results may occur due to:

• Varying endogenous FBP2 expression levels across cell types

• Different subcellular distributions of FBP2 (nuclear vs. cytoplasmic vs. mitochondrial)

• Presence of different oligomeric forms (dimers vs. tetramers)

• Metabolic state of cells influencing FBP2 localization and function

• Context-dependent functions of FBP2 (e.g., different effects under normoxia vs. hypoxia)

To address these issues:

• Validate antibody specificity using appropriate controls

• Perform careful subcellular fractionation when needed

• Consider the metabolic state of your cells (normal vs. stressed)

• Account for differences between cell types in FBP2 expression and regulation

What are common pitfalls when studying FBP2 in cancer models?

When investigating FBP2 in cancer contexts:

• Consider that FBP2 is silenced in many sarcoma subtypes, requiring restoration models rather than knockdown approaches

• Ensure FBP2 expression levels in engineered cells are comparable to appropriate control cells (e.g., HSMM for liposarcoma)

• Account for differential effects in various microenvironmental conditions (normoxia vs. hypoxia)

• Be aware that FBP2's functions may differ between cancer types (protective in some contexts, tumor-suppressive in others)

• When using doxycycline-inducible systems, carefully titrate doxycycline concentration to achieve physiologically relevant expression levels

How can I determine if my FBP2 antibody can detect both dimeric and tetrameric forms?

To evaluate antibody recognition of different oligomeric forms:

• Use purified recombinant FBP2 (wild-type and mutants like L190G and D187L)

• Subject proteins to conditions favoring different oligomeric states (varying AMP concentrations)

• Analyze by native PAGE followed by western blotting

• Compare detection efficiency across different oligomeric states

• If possible, analyze epitope accessibility in different oligomeric conformations based on structural data