PFKL Antibody

What is PFKL and why is it important in cellular metabolism?

PFKL is a rate-limiting enzyme in glycolysis that catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, representing the first committing step of glycolysis . It plays a crucial role in controlling cellular energy metabolism and cooperates with other enzymes like hexokinase and pyruvate kinase to ensure smooth conversion of glucose to pyruvate, facilitating subsequent processes like the citric acid cycle . Beyond its metabolic role, PFKL has been found to negatively regulate phagocyte oxidative burst in response to bacterial infection by controlling cellular NADPH biosynthesis and NADPH oxidase-derived reactive oxygen species. Additionally, upon macrophage activation, PFKL drives the metabolic switch toward glycolysis, thus preventing glucose turnover that produces NADPH via the pentose phosphate pathway .

What types of PFKL antibodies are available for research applications?

Based on the search results, several types of PFKL antibodies are available for research:

• Monoclonal antibodies: Such as mouse IgG1 monoclonal antibodies (e.g., 68385-1-Ig)

• Polyclonal antibodies: Including rabbit IgG polyclonal antibodies (e.g., 15652-1-AP)

• Phospho-specific antibodies: Antibodies specifically targeting phosphorylated Ser775 of PFKL

• Total PFKL antibodies: Antibodies that detect PFKL regardless of phosphorylation status

Each type has specific advantages depending on the research application. Monoclonal antibodies provide high specificity for a single epitope, while polyclonal antibodies can offer better detection sensitivity by recognizing multiple epitopes. Phospho-specific antibodies are crucial for studying post-translational modifications that affect PFKL activity.

What species cross-reactivity should be considered when selecting a PFKL antibody?

When selecting a PFKL antibody, researchers should carefully evaluate species cross-reactivity to ensure compatibility with their experimental models. The data shows variations in species reactivity among available antibodies:

It's critical to verify reactivity with your specific experimental model, especially when working with less common species. Some antibodies show broader cross-reactivity than others, making them more versatile for comparative studies across different animal models. The search results indicate that antibody 68385-1-Ig has been positively tested with multiple species including pig, rabbit, rat, and mouse brain tissues .

What are the validated applications for PFKL antibodies?

PFKL antibodies have been validated for multiple research applications with varying degrees of optimization required:

For example, antibody 68385-1-Ig has been positively detected in Western blot using various cell lines (LNCaP, HeLa, HEK-293, Jurkat, K-562) and tissue samples (pig, rabbit, rat, mouse brain tissues) . This wide validation across different sample types suggests high versatility for different experimental setups.

What are the recommended dilutions for different applications of PFKL antibodies?

Optimal dilutions vary significantly based on the specific application and antibody used:

These recommendations should serve as starting points, and researchers should titrate antibodies in their specific systems to achieve optimal results. The wide range of dilutions for Western blot (1:500 to 1:50000) highlights the importance of optimization for each experimental setup and sample type .

What sample preparation methods optimize PFKL detection in Western blotting?

For optimal PFKL detection in Western blotting, the search results suggest the following methodology:

• Protein extraction: Use RIPA lysis buffer with protease inhibitors like PMSF for tissue and cell protein extraction . This combination efficiently extracts and preserves PFKL protein integrity.

• Protein quantification: Measure protein concentration using a spectrophotometer to ensure consistent loading .

• Protein loading: Load approximately 30-60 μg of protein per lane for optimal detection. For example, 30 μg of HepG2 whole cell lysate was used successfully with antibody ab97443 , while 60 μg of protein was used in other studies .

• SDS-PAGE conditions: Use 7.5-12% gels depending on the desired resolution range. 7.5% SDS-PAGE has been successfully used with ab97443 , while 10-12% gels were used in other studies .

• Transfer conditions: Transfer to PVDF membrane (specific transfer buffer not specified in the search results but standard Towbin buffer or similar would be appropriate) .

• Blocking: Block the membrane with appropriate blocking solution (e.g., commercial blocking buffer) for approximately 10 minutes at room temperature.

• Expected molecular weight: Look for a band at approximately 85 kDa, which is the observed molecular weight of PFKL .

How can researchers distinguish between total PFKL and phosphorylated PFKL in experiments?

To distinguish between total PFKL and phosphorylated PFKL (specifically at Ser775), researchers should use phospho-specific antibodies in parallel with total PFKL antibodies:

• Generate or obtain phospho-specific antibodies: As described in the search results, monoclonal antibodies can be raised against a C-terminal PFKL peptide specifically containing phosphorylated Ser775 . These antibodies generate a specific signal to Ser775 phosphorylated PFKL in immunoblots.

• Use total PFKL antibodies: In parallel, use antibodies that recognize PFKL regardless of its phosphorylation state .

• Conduct parallel immunoblots: Run identical samples on two separate blots or strip and reprobe the same blot with both antibody types.

• Use appropriate controls: Include samples with known phosphorylation status. For example, resting macrophages (mBMDM) showed weak phospho-PFKL signal, while treatment with TLR ligands (LPS, R848, Pam₃CSK₄) increased phosphorylation .

• Validation using phosphorylation-defective mutants: Include samples expressing PFKL S775A mutants (where Ser775 is substituted with alanine) as negative controls for phospho-specific antibodies .

This approach enables researchers to monitor both the total expression levels of PFKL and its phosphorylation status, providing insights into its regulation under different experimental conditions.

What controls should be included when studying PFKL phosphorylation?

When studying PFKL phosphorylation, particularly at Ser775, the following controls are essential:

• Negative controls:

  • Unstimulated/resting cells: Resting macrophages show minimal PFKL Ser775 phosphorylation

  • Phosphorylation-defective mutants: PFKL S775A mutant where serine is replaced with alanine

  • Cells treated with relevant kinase inhibitors: Inhibitors of the kinases responsible for PFKL phosphorylation

• Positive controls:

  • Stimulated cells: Macrophages treated with TLR ligands (LPS, R848, Pam₃CSK₄) or other innate immune stimuli

  • In vitro phosphorylated PFKL: Recombinant PFKL treated with purified PKCδ shows dose-dependent phosphorylation at Ser775

• Loading controls:

  • Housekeeping proteins: β-actin has been successfully used as a loading control in PFKL studies

  • Total PFKL detection: When studying phosphorylation, parallel detection of total PFKL ensures changes reflect phosphorylation rather than expression

• Quantification controls:

  • Standard curves: To estimate the percentage of phosphorylated PFKL in samples. In one study, approximately 30% of wild-type PFKL enzyme was found to be phosphorylated under heterologous expression conditions

How can researchers assess PFKL enzymatic activity in relation to its phosphorylation state?

To assess how phosphorylation affects PFKL enzymatic activity, researchers can employ several complementary approaches:

• In vitro enzymatic assays:

  • Measure F1,6BP production over time using liquid chromatography-mass spectrometry

  • Compare initial reaction rates between wild-type PFKL and phosphorylation-defective mutants (e.g., PFKL S775A)

  • Research has shown that the enzymatic activity of the PFKL S775A mutant was approximately half of its wild-type counterpart

• Quantification of phosphorylated fraction:

  • Conduct immunoprecipitation experiments using phospho-specific antibodies

  • Determine the proportion of phosphorylated enzyme in your experimental system

  • In one study, approximately 30% of the wild-type PFKL enzyme was found to be phosphorylated under heterologous expression conditions

• Cellular glycolysis monitoring:

  • Use cells expressing phosphorylation-defective PFKL variants

  • Monitor glycolytic activity using techniques such as extracellular acidification rate (ECAR) measurements

  • Macrophages from genetic mouse models where PFKL Ser775 phosphorylation cannot occur show lower glycolysis upon activation compared to wild-type animals

• Downstream metabolic effects:

  • Assess downstream metabolic markers such as HIF1α levels

  • Monitor cytokine production (e.g., IL-1β) that depends on glycolytic activity

  • Wild-type cells with higher glycolytic activity have shown higher levels of HIF1α and IL-1β than cells expressing phosphorylation-defective PFKL

These approaches collectively provide a comprehensive understanding of how phosphorylation regulates PFKL activity and its impact on cellular metabolism.

How can site-specific crosslinking be used to study PFKL protein interactions and self-assembly?

Site-specific crosslinking offers a powerful approach to investigating PFKL's dynamic interactions and self-assembly properties:

• Incorporation of photochemical crosslinkers:

  • Site-specifically incorporate the photochemical crosslinking unnatural amino acid 4-azido-phenylalanine (AzF) into PFKL using amber stop codon (TAG) substitution technology

  • Target positions that lie at interfaces of PFKL filaments, such as K90 and H211 (near active site), K356 and Q359 (near interface 2), and K397, Y487, Y514, and V699 (near interface 1)

• Expression in cellular systems:

  • Co-transfect PFKL amber stop codon constructs with AzF orthogonal tRNA/tRNA synthetase into cells (e.g., HEK293T)

  • This system allows for the production of PFKL with site-specific crosslinking capability

• UV-induced crosslinking:

  • Expose cells to UV light to activate the crosslinker

  • This creates covalent bonds between PFKL and interacting proteins

  • Different interfaces show distinct crosslinking patterns, as evidenced by different band shifting patterns upon UV exposure

• Proteomic analysis of crosslinked products:

  • Use multiplexed TMT labeled affinity purification mass spectrometry (AP-MS) to quantitatively compare protein interactions across different PFKL interfaces

  • Interface 2 showed robust crosslinking with cytoskeletal proteins, revealing it as a hub for protein-protein interactions

• Investigation of PFKL inhibition dynamics:

  • Apply this technique under different conditions, such as citrate-induced inhibition

  • Research has shown that PFKL forms puncta under citrate-induced inhibition in human cells

This approach provides unique insights into PFKL's structural dynamics and interactome that would be difficult to capture using conventional protein interaction methods.

What signaling pathways regulate PFKL phosphorylation at Ser775?

PFKL phosphorylation at Ser775 is regulated by several signaling pathways, particularly in the context of innate immune responses:

• TLR signaling pathway:

  • Various Toll-like receptor (TLR) ligands induce PFKL Ser775 phosphorylation in macrophages

  • These include LPS (TLR4 ligand), R848 (TLR7/8 ligand), and Pam₃CSK₄ (TLR1/2 ligand)

  • The phosphorylation occurs downstream of the IKK complex but independent of AKT signaling

• C-type lectin receptor signaling:

  • Dectin-1 stimulation also leads to PFKL phosphorylation at Ser775

  • In this context, PKCδ is required for the phosphorylation event

• Direct kinase regulation:

  • PKCδ has been identified as a direct kinase for PFKL Ser775

  • In vitro kinase assays with recombinant PKCδ and human PFKL showed dose-dependent phosphorylation of wild-type PFKL at Ser775

  • No phosphorylation was detected for the PFKL S775A mutant, confirming specificity

• Computational predictions:

  • Kinase prediction tools ranked PKCδ high (9th for mouse PFKL Ser775 site and 15th for human PFKL Ser775 site) among 303 kinases considered

Understanding these regulatory pathways is crucial for interpreting PFKL's role in metabolic adaptation during immune responses and may provide insights into potential therapeutic targets for metabolic and inflammatory diseases.

How does PFKL phosphorylation impact innate immune responses and metabolic reprogramming?

PFKL phosphorylation at Ser775 serves as a critical link between innate immune signaling and metabolic adaptation:

• Enhanced glycolytic activity:

  • Phosphorylation at Ser775 increases PFKL's catalytic activity

  • Enzymatic assays showed that phosphorylation-defective PFKL S775A mutant had approximately half the activity of wild-type PFKL

  • This leads to increased glycolysis in activated macrophages

• Metabolic shift during immune activation:

  • Upon macrophage activation, PFKL phosphorylation drives a metabolic switch toward glycolysis

  • This prevents glucose turnover through the pentose phosphate pathway

  • The shift is critical for supporting the energy demands of activated immune cells

• Regulation of cytokine production:

  • Enhanced glycolysis in wild-type cells correlates with higher levels of HIF1α and IL-1β compared to cells with phosphorylation-defective PFKL

  • This suggests that PFKL phosphorylation influences inflammatory cytokine production by modulating cellular metabolism

• Genetic evidence:

  • A genetic mouse model in which PFKL Ser775 phosphorylation cannot occur showed lower glycolysis in macrophages upon activation compared to wild-type animals

  • This confirms the physiological relevance of this phosphorylation event in vivo

• Broader implications:

  • Given PFKL's expression pattern, the activation through phosphorylation likely extends beyond innate immune cells

  • This suggests a broader role in metabolic adaptation across different cell types and physiological contexts

This research highlights PFKL phosphorylation as a proximal signaling event connecting innate immune responses to metabolic reprogramming, providing insights into how cells rapidly adapt their metabolism to meet the energetic demands of immune activation.

What role does PFKL play in disease processes and potential therapeutic targeting?

PFKL has been implicated in various disease processes, particularly those involving metabolic dysregulation:

• Cancer metabolism:

  • PFKL has been studied in hepatoma cells, where it appears to influence signaling pathways beyond its metabolic function

  • Research has shown that PFKL knockdown not only reduced RPIA (ribose 5-phosphate isomerase A) levels but also decreased phosphorylated ERK levels without affecting phosphorylated Raf

  • PFKL overexpression reduced PP2A activity in hepatoma cell lines, similar to the effect of RPIA overexpression

  • These findings suggest PFKL may contribute to cancer cell signaling and survival through mechanisms beyond glycolysis

• Inflammatory diseases:

  • Given PFKL's role in innate immune responses and metabolic reprogramming in macrophages , it may be implicated in inflammatory diseases

  • Targeting PFKL phosphorylation could potentially modulate inflammatory responses by affecting metabolic adaptation in immune cells

• ApoM-related disorders:

  • ApoM gene knockout significantly increased the expression levels of SREBF1 and PFKL

  • This suggests a link between lipoprotein metabolism and glycolytic regulation that may be relevant to metabolic disorders

• Potential therapeutic approaches:

  • Inhibiting PFKL phosphorylation: Developing compounds that prevent Ser775 phosphorylation could potentially modulate glycolytic activity in disease contexts

  • Targeting PFKL protein-protein interactions: The identification of interface 2 as a hub for protein interactions suggests potential for disrupting specific interactions

  • Modulating PFKL self-assembly: PFKL forms puncta under citrate-induced inhibition , suggesting that targeting its assembly dynamics could affect its function

Future research should further elucidate PFKL's role in specific disease contexts and explore its potential as a therapeutic target for metabolic and inflammatory disorders.

What emerging techniques are advancing PFKL research beyond traditional antibody applications?

Several cutting-edge techniques are advancing PFKL research beyond conventional antibody applications:

• Site-specific incorporation of unnatural amino acids:

  • Incorporation of photo-chemical crosslinking unnatural amino acid azido-phenylalanine (AzF) allows precise study of PFKL interactions

  • This technique enables investigation of specific interfaces and domains within the PFKL protein

• TMT multiplexed proteomics:

  • Tandem Mass Tag (TMT) multiplexed proteomics combined with site-specific crosslinking provides quantitative analysis of the PFKL interactome

  • This approach revealed interface 2 as a hub for protein-protein interactions, particularly with cytoskeletal proteins

• In vitro reconstitution of PFKL activity:

  • Recombinant PFKL production and enzymatic assays enable detailed biochemical characterization

  • Liquid chromatography-mass spectrometry methods for measuring F1,6BP production provide sensitive and quantitative assessment of PFKL activity

• Genetic mouse models:

  • Development of mice in which PFKL Ser775 phosphorylation cannot occur allows investigation of physiological relevance in vivo

  • These models enable study of metabolic reprogramming in primary cells under physiologically relevant conditions

• Advanced microscopy techniques:

  • Visualization of PFKL puncta formation under citrate-induced inhibition

  • This suggests the potential for using super-resolution microscopy to further investigate PFKL self-assembly and localization

• CRISPR-mediated genome editing:

  • While not explicitly mentioned in the search results, CRISPR technology enables precise modification of PFKL at the genomic level

  • This allows study of PFKL variants in endogenous contexts without overexpression artifacts

These emerging techniques are providing unprecedented insights into PFKL structure, function, regulation, and interactions, promising to deepen our understanding of this key metabolic enzyme and its diverse roles in cellular physiology.