Phospho-CASP9 (S144) Antibody

Basic Research Questions

• What is Phospho-CASP9 (S144) Antibody and how does it function in experimental contexts?

Phospho-CASP9 (S144) antibody is a polyclonal antibody specifically designed to detect endogenous levels of caspase-9 protein only when phosphorylated at Serine 144. This antibody is typically developed by immunizing rabbits with synthetic phosphopeptides derived from human CASP9 surrounding the Ser144 phosphorylation site . The antibody functions through specific epitope recognition, binding exclusively to the phosphorylated form while showing no reactivity with the non-phosphorylated protein. This specificity makes it an invaluable tool for studying post-translational modifications that regulate apoptotic pathways .

• What are the validated applications for Phospho-CASP9 (S144) Antibody?

The Phospho-CASP9 (S144) antibody has been validated for multiple research applications with specific working dilutions:

These applications have been validated with human samples, with some antibodies also demonstrating reactivity with mouse and rat samples . Western blot analysis has confirmed detection in multiple cell lines including HEK293T, HepG2, and A375 whole cell lysates at approximately 47 kDa .

• How should Phospho-CASP9 (S144) Antibody be properly stored and handled?

Proper storage and handling are critical for maintaining antibody functionality:

• Short-term storage: 4°C (up to several weeks)

• Long-term storage: -20°C (stable for approximately one year after receipt)

• Aliquoting is essential to avoid repeated freeze-thaw cycles which can degrade antibody performance

• Typical storage buffer composition: PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

• Working dilutions should be prepared fresh before use

Following these guidelines ensures optimal antibody performance and specificity while preventing degradation that could compromise experimental results .

• What is the molecular function of Caspase-9 and its role in the apoptotic pathway?

Caspase-9 (CASP9) is a critical initiator caspase in the intrinsic (mitochondrial) apoptotic pathway. The functional mechanism involves:

• Initial form: 47 kDa procaspase-9 zymogen with minimal activity

• Activation trigger: Cytochrome c release from mitochondria following apoptotic stimuli

• Complex formation: Procaspase-9 associates with Apaf-1 (Apoptotic protease-activating factor 1) to form the apoptosome

• Proteolytic processing: Cleavage occurs at Asp315, producing a p35 subunit

• Secondary cleavage: Further processing at Asp330 produces a p37 subunit that amplifies the apoptotic response

• Effector activation: Cleaved caspase-9 processes downstream caspases (primarily caspase-3 and caspase-7)

• Outcome: Initiation of the caspase cascade leading to cellular apoptosis

This pathway represents a fundamental mechanism for programmed cell death that removes damaged or unnecessary cells during development and tissue homeostasis .

Intermediate Research Questions

• How does phosphorylation at Serine 144 regulate Caspase-9 function?

Phosphorylation at Serine 144 serves as an inhibitory regulatory mechanism for Caspase-9:

• Functional effect: Inhibits caspase-9 activation and subsequent caspase-3 activation

• Cellular outcome: Restrains the intrinsic apoptotic pathway, promoting cell survival

• Experimental validation: Prevention of Ser144 phosphorylation (through PKCζ inhibition or caspase-9 mutation) promotes caspase-3 activation and apoptosis

• Specificity: This phosphorylation represents a distinct regulatory mechanism from other known phosphorylation sites on caspase-9

• Structural implications: Likely alters protein conformation to prevent proper apoptosome formation or substrate interaction

This post-translational modification represents one of several mechanisms by which survival signaling pathways can impinge on the apoptotic machinery to maintain cellular viability during specific stress conditions .

• Which kinases phosphorylate Caspase-9 at Serine 144 and under what conditions?

The primary kinase responsible for phosphorylating Caspase-9 at Serine 144 is protein kinase C zeta (PKCζ):

• Kinase identification: Determined through inhibitor sensitivity profiles and direct protein-protein interaction studies

• Kinase classification: PKCζ belongs to the atypical PKC subfamily (insensitive to both diacylglycerol and Ca²⁺)

• Activation conditions:

  • Treatment with protein phosphatase inhibitors (e.g., okadaic acid)

  • Hyperosmotic stress (specifically activates PKCζ)

• Selectivity: Phosphorylation at Ser144 is not induced by growth factors, phorbol esters, or other cellular stresses

• Functional context: Part of the survival response during hyperosmotic challenge

This specific kinase-substrate relationship provides a mechanistic link between osmotic stress response pathways and apoptotic regulation .

• How can researchers induce and detect Caspase-9 phosphorylation at Ser144 in experimental systems?

To effectively study Ser144 phosphorylation, researchers can employ several experimental strategies:

Induction methods:

• Treat cells with okadaic acid (protein phosphatase inhibitor)

• Subject cells to hyperosmotic stress conditions

• Express constitutively active PKCζ constructs

Detection methods:

• Western blotting with Phospho-CASP9 (S144) antibody (1:500-1:2000 dilution)

• Immunofluorescence to visualize cellular localization (1:50-1:200 dilution)

• Immunohistochemistry for tissue sections (1:100-1:300 dilution)

Controls for validation:

• Use PKCζ inhibitors as negative controls

• Employ S144A mutant caspase-9 (non-phosphorylatable) for specificity validation

• Include both phosphorylated and non-phosphorylated samples to confirm antibody specificity

These methodological approaches enable systematic investigation of this regulatory mechanism in diverse experimental contexts .

• How does Caspase-9 phosphorylation at Ser144 compare with other known phosphorylation sites?

Caspase-9 is one of the most extensively phosphorylated caspases, with at least 11 distinct phosphorylation sites across all domains. Key comparative insights include:

*While specific kinases for Ser196 weren't identified in the search results, commercial antibodies target this site .

Caspase-9 thus serves as a focal point for multiple protein kinase signaling pathways that regulate apoptosis, with each phosphorylation site potentially integrating different cellular signals .

Advanced Research Questions

• What are the mechanistic differences between Ser144 phosphorylation and other inhibitory modifications of Caspase-9?

The inhibitory phosphorylation of Caspase-9 at different sites represents distinct regulatory mechanisms with specific physiological contexts:

• Ser144 phosphorylation by PKCζ:

  • Specifically induced by hyperosmotic stress

  • Not responsive to growth factors or phorbol esters

  • Represents a targeted stress-response mechanism

  • Appears linked to cellular volume regulation pathways

• Thr125 phosphorylation by ERK:

  • Regulated by growth factor signaling pathways

  • Commonly upregulated in cancer cells

  • May contribute to apoptotic resistance in tumors

  • Integrated with proliferative signaling networks

• Thr125 phosphorylation by CDK1:

  • Cell cycle-dependent regulation

  • Specifically functions during mitosis

  • Prevents inappropriate apoptosis during cell division

  • Coordinated with cell cycle progression

These mechanistic differences illustrate how multiple signaling pathways converge on Caspase-9 to integrate diverse cellular conditions into a coherent apoptotic decision .

• What experimental controls are essential when using Phospho-CASP9 (S144) antibody in complex experimental systems?

Robust experimental design with appropriate controls is critical when using phospho-specific antibodies:

Positive Controls:

• Cells treated with okadaic acid

• Cells subjected to hyperosmotic stress

• Lysates from cells overexpressing constitutively active PKCζ

Negative Controls:

• Samples treated with λ-phosphatase to remove all phosphorylations

• Cells expressing S144A mutant caspase-9 (cannot be phosphorylated)

• Samples from cells treated with PKCζ inhibitors

Specificity Controls:

• Peptide competition assays (phosphorylated vs. non-phosphorylated peptides)

• Dual labeling with total CASP9 antibody to normalize signals

• CASP9 knockdown/knockout cells to confirm signal specificity

Application-Specific Controls:

• For Western blots: Include molecular weight markers (expect ~47 kDa band)

• For IHC/IF: Include isotype control antibodies

• For quantitative applications: Generate standard curves with known quantities of phosphorylated protein

These controls ensure that observed signals truly represent Ser144 phosphorylation status rather than experimental artifacts .

• How can Phospho-CASP9 (S144) antibody be incorporated into multi-parameter apoptosis studies?

For comprehensive apoptosis research, Phospho-CASP9 (S144) antibody can be integrated into multi-parameter studies:

Sequential activation analysis:

• Combine with antibodies detecting total CASP9, cleaved CASP9 (Asp315), cleaved CASP3, and PARP cleavage

• Create temporal profiles of phosphorylation status relative to cleavage events

• Correlate phosphorylation with inhibition of downstream apoptotic events

Pathway integration studies:

• Pair with analysis of PKCζ activation (phospho-PKCζ detection)

• Include markers for mitochondrial outer membrane permeabilization

• Measure cytochrome c release in parallel with caspase phosphorylation

Single-cell analysis approaches:

• Multiplex immunofluorescence to correlate CASP9 phosphorylation with other apoptotic markers

• Flow cytometry to quantify phospho/total CASP9 ratios across different cell populations

• Live-cell imaging with appropriate reporters to track phosphorylation dynamics

Stress-response profiling:

• Compare phosphorylation patterns across different stress conditions

• Correlate with cell survival metrics and recovery capacity

• Examine temporal dynamics of phosphorylation/dephosphorylation cycles

These approaches provide mechanistic insight into how phosphorylation regulates apoptotic execution in diverse physiological contexts .

• What are the implications of Caspase-9 Ser144 phosphorylation for cancer research and therapeutic development?

Phosphorylation of Caspase-9 at Ser144 has significant implications for cancer biology and therapy:

Cancer resistance mechanisms:

• Increased Ser144 phosphorylation may contribute to apoptotic resistance in tumor cells

• PKCζ dysregulation could represent an unrecognized mechanism of therapy evasion

• Monitoring phosphorylation status could potentially predict treatment response

Therapeutic targeting opportunities:

• PKCζ inhibitors might sensitize resistant tumors to apoptosis-inducing therapies

• Combination approaches targeting multiple phosphorylation sites could overcome compensatory mechanisms

• Development of compounds that specifically block the interaction between PKCζ and Caspase-9

Biomarker development:

• Phospho-CASP9 (S144) levels could serve as indicators of specific resistance mechanisms

• Ratio of phosphorylated to total CASP9 might predict therapeutic vulnerability

• Changes in phosphorylation status during treatment could indicate adaptive responses

Research applications:

• Screening for compounds that modulate Ser144 phosphorylation

• Understanding the relationship between osmotic stress and apoptotic regulation in tumor microenvironments

• Identifying patient subgroups that might benefit from PKCζ-targeting approaches

These research directions highlight the potential translational significance of this phosphorylation site in cancer treatment strategies .

• How does osmotic stress regulate the PKCζ-mediated phosphorylation of Caspase-9?

The mechanism connecting hyperosmotic stress to Caspase-9 phosphorylation involves several coordinated steps:

Stress sensing pathway:

• Hyperosmotic conditions trigger cellular volume changes

• Osmosensors (likely membrane proteins) detect these alterations

• Signal transduction cascades activate PKCζ through mechanisms distinct from classical PKC activation

PKCζ activation mechanism:

• Unlike conventional PKCs, PKCζ is insensitive to Ca²⁺ and diacylglycerol

• Activation likely involves protein-protein interactions and/or phosphorylation events

• Activated PKCζ specifically interacts with Caspase-9, as demonstrated by co-immunoprecipitation studies

Kinase-substrate interaction:

• Activated PKCζ binds to Caspase-9

• Phosphorylation occurs specifically at Ser144

• This modification prevents Caspase-9 activation

• The apoptotic pathway is restrained during osmotic adaptation

Pathway specificity:

• Other cellular stresses (oxidative, genotoxic, ER stress) do not trigger this phosphorylation

• Growth factors and phorbol esters (which activate other PKC isoforms) do not induce Ser144 phosphorylation

• This represents a highly specific stress-response mechanism

Understanding this pathway provides insight into how cells maintain survival during osmotic challenges frequently encountered in physiological contexts .

• What methodological approaches can be used to study the dynamics of Caspase-9 phosphorylation in live cells?

While standard phospho-antibodies cannot be used in live cells, several advanced approaches can be employed to study phosphorylation dynamics:

Genetically encoded biosensors:

• Design FRET-based reporters incorporating the Ser144 region of Caspase-9

• Changes in FRET signal occur upon phosphorylation/dephosphorylation

• Enables real-time visualization of phosphorylation status

Engineered expression systems:

• Create cell lines stably expressing fluorescently-tagged wild-type and S144A Caspase-9

• Compare localization and protein interaction dynamics

• Measure differential apoptotic responses to stimuli

Temporal analysis:

• Conduct time-course experiments with fixation and immunostaining

• Generate kinetic profiles of phosphorylation under different conditions

• Correlate with other cellular events (e.g., PKCζ activation, apoptosis markers)

Advanced microscopy:

• Apply techniques like fluorescence correlation spectroscopy to study molecular interactions

• Use super-resolution microscopy to examine spatial organization of signaling complexes

• Implement light-sheet microscopy for extended live imaging with reduced phototoxicity

These approaches enable researchers to move beyond static snapshots toward understanding the dynamic regulation of Caspase-9 in intact cellular systems .

• How can phosphorylation of Caspase-9 at multiple sites be integrated into systems biology models of apoptosis?

Integrating multiple phosphorylation events into systems biology models requires sophisticated approaches:

Quantitative phosphoproteomics:

• Measure absolute stoichiometry of phosphorylation at each site

• Determine site occupancy under different conditions

• Establish temporal relationships between modifications

Mathematical modeling:

• Develop differential equation models incorporating phosphorylation/dephosphorylation kinetics

• Include kinase activities, phosphatase actions, and downstream effects

• Simulate interaction between multiple regulatory sites

Network integration:

• Map connections between upstream signaling pathways and individual phosphorylation sites

• Model crosstalk and feedback mechanisms between survival and apoptotic pathways

• Predict cellular outcomes based on phosphorylation patterns

Experimental validation:

• Generate phosphomimetic and phospho-null mutations at multiple sites

• Conduct epistasis experiments to determine hierarchy of modifications

• Apply optogenetic approaches to achieve temporal control over specific kinases

Translational applications:

• Predict therapeutic vulnerabilities based on phosphorylation profiles

• Design rational drug combinations targeting multiple regulatory nodes

• Develop personalized treatment approaches based on patient-specific phosphorylation patterns