pi4kb Antibody

What is PI4KB and what cellular functions does it regulate?

PI4KB (phosphatidylinositol 4-kinase beta) is an essential enzyme that phosphorylates phosphatidylinositol (PI) in the first committed step of generating the second messenger inositol-1,4,5,-trisphosphate (PIP) . It belongs to the PI3/PI4-kinase family and is primarily localized at the Golgi and trans-Golgi network (TGN) .

PI4KB regulates several critical cellular processes:

• Golgi disintegration and reorganization during mitosis

• Golgi-to-plasma membrane trafficking

• Generation of PI4P pools in the Golgi apparatus

• Inner ear development

• Serves as a host factor in viral replication for multiple viruses

The protein has a calculated molecular weight of approximately 90 kDa but is commonly observed at around 100 kDa in Western blots .

What applications can PI4KB antibodies be used for?

PI4KB antibodies have been validated for multiple experimental applications:

For all applications, it's recommended to optimize antibody concentration for specific experimental conditions .

What are recommended sample preparation techniques for PI4KB detection?

For optimal PI4KB detection:

• Western Blot: Lyse cells in RIPA buffer with protease inhibitors. PI4KB is observed at approximately 100 kDa. Successfully detected in K-562 cells, NIH-3T3 cells, and various human, mouse, and rat tissues .

• Immunohistochemistry: For paraffin-embedded tissues, antigen retrieval with TE buffer pH 9.0 is recommended. Alternatively, citrate buffer pH 6.0 can be used. Human liver tissue has shown positive detection .

• Immunofluorescence: C6 and HeLa cells have shown reliable detection. Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, permeabilize with 0.1% Triton X-100 .

• Positive control tissues/cells: K-562 cells, NIH-3T3 cells, and human liver tissue consistently show good results for validation experiments .

How should PI4KB antibodies be stored and handled for optimal performance?

Storage and handling recommendations for PI4KB antibodies:

• Storage temperature: Most PI4KB antibodies should be stored at -20°C for long-term storage .

• Storage buffer: Typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• Stability: Generally stable for one year after shipment when stored properly .

• Handling: Aliquoting is recommended for antibodies without glycerol to avoid freeze-thaw cycles. Antibodies in 50% glycerol do not typically require aliquoting for -20°C storage .

• After reconstitution: Lyophilized antibodies should be stored at 4°C for one month after reconstitution or aliquoted and stored at -20°C for six months .

• Avoid: Repeated freezing and thawing, which can reduce antibody performance .

How can PI4KB antibodies be utilized to investigate viral replication mechanisms?

PI4KB plays critical roles in the replication of several RNA viruses:

Enterovirus replication:

• PI4KB, in complex with c10orf76, generates PI4P at membranous replication organelles .

• Experimental approach: Use PI4KB antibodies to perform co-immunoprecipitation with viral proteins to identify interactions. PI4KB inhibitors like PIK93 (at 2-4 μM) can be used to validate functional roles .

• Viral minigenome assays have demonstrated that PI4KB increases reporter activity in a dose-dependent manner, directly supporting viral genome replication .

Human Parainfluenza Virus-3 (HPIV3):

• PI4KB is recruited to inclusion bodies (IBs) via interaction with the viral P protein .

• Methodological approach: Knockdown of PI4KB using shRNA significantly reduces HPIV3 replication, which can be rescued by expression of wild-type PI4KB but not by kinase-dead mutants .

• Immunofluorescence staining reveals PI4KB colocalizes with viral inclusion bodies, and this recruitment is mediated by the viral P protein rather than N protein .

SARS-CoV entry:

• PI4KB is required for cellular spike-mediated entry of human coronavirus SARS-CoV .

• Research approach: Combine PI4KB antibodies with viral entry assays to track PI4KB recruitment during viral infection.

What is the relationship between PI4KB and c10orf76 in cellular signaling pathways?

The PI4KB-c10orf76 complex represents a key regulatory mechanism in phosphoinositide signaling:

• Complex formation: c10orf76 (79 kDa) and PI4KB (89 kDa) form a 1:1 heterodimeric complex (158 kDa) as determined by size-exclusion chromatography .

• Binding mechanism: Hydrogen-deuterium exchange mass spectrometry revealed that binding is mediated by the kinase linker of PI4KB through a disorder-to-order transition .

• Regulation: Formation of the heterodimeric complex is modulated by PKA-dependent phosphorylation of the c10orf76 binding site on PI4KB .

• Functional significance:

  • Knockout of c10orf76 leads to decreased PI4P levels and disruption of Arf1 activation in cells

  • c10orf76 is recruited to the Golgi by PI4KB

  • The complex is essential for the replication of specific enteroviruses

Experimental approach: Use PI4KB antibodies for co-immunoprecipitation with c10orf76 to study complex formation under different cellular conditions or mutations. Western blotting can confirm complex formation while immunofluorescence can visualize colocalization at the Golgi.

How can researchers distinguish between different PI4K isoforms in their experiments?

Four PI4K isoforms exist in humans: PI4KIIα (PI4K2A), PI4KIIβ (PI4K2B), PI4KIIIα (PI4KA), and PI4KIIIβ (PI4KB) . Distinguishing between them requires specific approaches:

Antibody selection:

• Use isoform-specific antibodies that have been validated against knockout/knockdown controls

• When performing immunofluorescence, compare localization patterns with known markers (PI4KB is primarily Golgi-localized)

Functional discrimination:

• Isoform-specific inhibitors: PIK93 at 2-4 μM is selective for PI4KB

• Isoform-specific knockdown: shRNA targeting PI4KB specifically reduces viral replication dependent on this isoform

Localization patterns:

• PI4KB: Primarily Golgi and trans-Golgi network

• PI4KA: Not recruited to viral inclusion bodies

• PI4K2A: Not recruited to viral inclusion bodies

• PI4K2B: Can colocalize with inclusion bodies but knockdown doesn't affect viral replication

Research finding: In studies of HPIV3 viral inclusion bodies, only PI4KB knockdown significantly affected viral replication, although both PI4KB and PI4K2B colocalized with inclusion bodies .

What are the optimal experimental controls when working with PI4KB antibodies?

Essential controls for PI4KB antibody experiments:

For Western blot:

• Positive controls: K-562 cells, NIH-3T3 cells have shown reliable detection

• Negative controls:

  • RNAi-treated NIH-3T3 cells show reduced signal

  • Kinase-dead PI4KB mutants for functional studies

• Loading controls: Standard housekeeping proteins like GAPDH or β-actin

For immunoprecipitation:

• Input control: 5-10% of pre-IP lysate

• IgG control: Non-specific rabbit IgG

• Successfully validated in: K-562 cells

For immunofluorescence:

• Positive controls: C6 cells show reliable detection

• Subcellular markers: Co-stain with Golgi markers (GM130, TGN46)

• Specificity controls: PI4KB knockdown/knockout cells

• Functional controls: Wild-type vs. kinase-dead PI4KB shows recruitment without function

For genetic manipulation validation:

• Rescue experiments: Wild-type PI4KB restores function in knockdown cells while kinase-dead PI4KB does not

• Phenotypic validation: Monitor PI4P levels using PI4P-specific probes in conjunction with PI4KB manipulation

How does phosphorylation regulate PI4KB activity and how can this be investigated?

PI4KB activity is regulated by phosphorylation, particularly by PKA:

Regulatory mechanism:

• PKA-dependent phosphorylation modulates the formation of the PI4KB-c10orf76 complex by modifying the c10orf76 binding site on PI4KB

• This phosphorylation affects the affinity of the complex formation and subsequently PI4P production

Experimental approaches:

• Phospho-specific antibodies: While not specifically mentioned in the search results, researchers could develop or obtain phospho-specific antibodies against known PKA phosphorylation sites on PI4KB

• Pharmacological manipulation:

  • PKA activators (forskolin, cAMP analogs) to enhance phosphorylation

  • PKA inhibitors (H-89, PKI) to prevent phosphorylation

• Mutational analysis:

  • Generate phospho-mimetic (S/T to D/E) or phospho-deficient (S/T to A) mutants of PI4KB at PKA sites

  • Use PI4KB antibodies to immunoprecipitate these mutants and assess their binding to c10orf76

• Functional readouts:

  • Monitor PI4P levels using PI4P-specific probes or antibodies

  • Assess Golgi morphology and Arf1 activation as downstream effects

• Mass spectrometry:

  • Use hydrogen-deuterium exchange mass spectrometry to characterize conformational changes induced by phosphorylation

What role does PI4KB play in pathological conditions and how can antibodies help investigate these mechanisms?

Viral infections:

• Enteroviruses: PI4KB generates PI4P for viral replication organelles

• HPIV3: PI4KB is recruited to inclusion bodies via the viral P protein

• SARS-CoV: Required for spike-mediated viral entry

• Aichi virus: Essential for RNA replication, recruited by ACBD3 to viral replication sites

Investigative approaches:

• Colocalization studies: Use PI4KB antibodies to track recruitment to viral replication sites

• Protein-protein interactions: Immunoprecipitate PI4KB to identify viral binding partners

• Functional studies: Combine PI4KB antibodies with viral replication assays

• Therapeutic targeting: Use PI4KB antibodies to validate inhibitor specificity

Genetic disorders:

• May play a role in inner ear development, with implications for hearing disorders

• DFNA87 is associated with PI4KB (based on gene symbol in search results)

Experimental approaches:

• Expression analysis: Quantify PI4KB levels in affected tissues

• Localization studies: Examine PI4KB distribution in normal vs. pathological samples

• Mutation analysis: Assess effects of disease-associated mutations on PI4KB function

What are the recommended dilutions for different applications of PI4KB antibodies?

The optimal dilutions vary by application and specific antibody:

It is recommended to titrate each antibody in specific testing systems to obtain optimal results as sensitivity may be sample-dependent .

How can researchers troubleshoot weak or non-specific signals when using PI4KB antibodies?

For weak signals:

• Increase antibody concentration: Try higher concentrations within the recommended range

• Optimize protein loading: Increase sample amount for low-expressing tissues/cells

• Enhance detection: Use more sensitive detection systems (ECL Plus, fluorescent secondaries)

• Antigen retrieval: For IHC, try alternative buffers - TE buffer pH 9.0 or citrate buffer pH 6.0

• Blocking optimization: Try different blocking agents (BSA, milk, commercial blockers)

• Incubation time: Extend primary antibody incubation (overnight at 4°C)

For non-specific signals:

• Antibody dilution: Use higher dilutions to reduce background

• Blocking optimization: Increase blocking time or concentration

• Washing steps: Add additional or longer washing steps

• Validated controls: Include PI4KB knockdown/knockout samples

• Secondary antibody controls: Run a lane with secondary antibody only

• Cross-reactivity testing: Perform peptide competition assays with immunogen

Sample preparation considerations:

• Fresh samples typically yield better results than frozen samples

• For Western blot, RIPA buffer with protease inhibitors is recommended

• For immunofluorescence, 4% paraformaldehyde fixation for 15 minutes at room temperature

How can PI4KB antibodies be used to study protein-protein interactions in research?

PI4KB forms critical interactions with both host and viral proteins:

Methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Successfully used to demonstrate PI4KB interaction with viral P protein in HPIV3

  • Protocol: Lyse cells in non-denaturing buffer, immunoprecipitate with PI4KB antibody, and probe for interacting partners

  • Reverse Co-IP with partner protein antibodies confirms specificity

• Proximity Ligation Assay (PLA):

  • Can detect protein interactions in fixed cells with spatial resolution

  • Combine PI4KB antibody with antibody against suspected interactor

• FRET/BRET assays:

  • For live-cell analysis of protein interactions

  • Tag PI4KB and interacting proteins with appropriate fluorophores

Key PI4KB interactions identified:

• c10orf76: Forms 1:1 complex with PI4KB, regulates PI4P production at Golgi

• HPIV3 P protein: Recruits PI4KB to viral inclusion bodies

• ACBD3: Recruits PI4KB to Aichi virus replication sites

• Viral 3A protein: Multiple picornaviruses utilize 3A to recruit PI4KB

Research finding: While both PI4KB and the viral N protein interact with the viral P protein in HPIV3, N interacts with PI4KB only in the presence of P, forming a tripartite complex essential for viral replication .

How are PI4KB antibodies being used in emerging viral research?

Recent research has expanded our understanding of PI4KB's role in viral infections:

SARS-CoV entry mechanism:

• PI4KB has been identified as required for cellular spike-mediated entry of SARS-CoV

• Research approach: Combine PI4KB antibodies with viral entry assays to dissect the mechanism

Enterovirus replication complexes:

• The c10orf76-PI4KB complex is essential for replication of specific enteroviruses

• Technical approach: Use complex-disrupting mutations to define the role of c10orf76-PI4KB in viral replication

HPIV3 inclusion bodies:

• PI4KB generates PI4P on viral inclusion bodies, facilitating viral replication

• Experimental strategy: PI4KB inhibition with PIK93 at 2-4 μM reduces PI4P generation and viral replication

Research finding: Kinase-dead PI4KB is still recruited to viral replication sites but cannot generate PI4P, indicating recruitment and function can be separated . This suggests targeting the kinase activity rather than recruitment might be more effective for antiviral strategies.

What is the significance of PI4KB in phosphoinositide metabolism research?

PI4KB plays a crucial role in phosphoinositide metabolism and signaling:

Key functions in phosphoinositide pathways:

• Phosphorylates phosphatidylinositol (PI) to generate PI4P at the Golgi

• PI4P serves as a precursor for PI(4,5)P₂ and subsequently PI(3,4,5)P₃

• Regulates Golgi-to-plasma membrane trafficking

Research approaches:

• PI4P pool quantification:

  • PI4P-specific antibodies or biosensors to measure PI4P levels

  • Compare PI4P levels in control vs. PI4KB knockdown/inhibited cells

• Membrane trafficking studies:

  • Cargo trafficking assays from Golgi to plasma membrane

  • Combine with PI4KB manipulation to establish causality

• Arf1 activation:

  • PI4KB and c10orf76 regulate Arf1 activation

  • Use Arf1 activation assays as functional readouts of PI4KB activity

Research finding: Knockout of c10orf76, a PI4KB interactor, leads to decreased PI4P levels and disruption of Arf1 activation in cells, demonstrating the importance of PI4KB complexes in regulating both phosphoinositide levels and downstream effectors .

How do researchers evaluate potential off-target effects when using PI4KB antibodies?

Comprehensive validation approaches:

• Genetic controls:

  • CRISPR knockout/knockdown: Compare antibody signals in wild-type vs. PI4KB-depleted samples

  • Overexpression: Verify increased signal with PI4KB overexpression

  • Recovery experiments: Restore signal by expressing RNAi-resistant PI4KB in knockdown cells

• Specificity controls:

  • Cross-reactivity testing: Verify antibody doesn't detect related PI4K family members

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

  • Multiple antibodies: Use antibodies targeting different epitopes of PI4KB to confirm results

• Technical controls:

  • Secondary-only controls: Ensure secondary antibodies don't contribute to signal

  • Isotype controls: Use matched isotype control antibodies

  • Titration: Ensure signal changes proportionally with antibody concentration

• Functional validation:

  • Structure-function studies: Compare wild-type vs. kinase-dead PI4KB

  • Inhibitor studies: Confirm phenotypic effects using PI4KB-specific inhibitors like PIK93