Phospho-RB1 (T821) Antibody

What is the biological significance of RB1 T821 phosphorylation?

The phosphorylation of RB1 at threonine 821 (T821) plays a crucial role in regulating the tumor suppressor function of the retinoblastoma protein. T821 is one of 15 potential CDK phosphorylation sites on RB1, and its phosphorylation status directly affects RB1's interaction with E2F transcription factors . When T821 is phosphorylated by cyclin-dependent kinases (CDKs), this modification contributes to the dissociation of RB1 from E2F, thereby enabling transcription of E2F-responsive genes and promoting cell cycle progression through the G1/S transition .

How does T821 phosphorylation differ from other RB1 phosphorylation sites?

T821 phosphorylation has distinctive characteristics compared to other RB1 phosphorylation sites:

This site-specific phosphorylation pattern suggests that T821 plays a unique role in RB1-mediated cellular responses, particularly in stress conditions that may trigger apoptosis .

What are the recommended applications for Phospho-RB1 (T821) antibodies?

Phospho-RB1 (T821) antibodies have been validated for multiple research applications, with specific protocols optimized for each technique:

For all applications, it is essential to include appropriate controls, including both positive samples (proliferating cells with phosphorylated RB1) and negative controls (such as phosphopeptide competition assays) .

How can I distinguish between mono-phosphorylated and hyper-phosphorylated RB1 in my experiments?

Distinguishing between mono-phosphorylated and hyper-phosphorylated RB1 requires careful experimental design:

Recommended methodological approach:

• Two-dimensional isoelectric focusing (2D IEF): This technique can separate RB1 protein based on both molecular weight and charge, allowing visualization of different phosphorylation states. Mono-phosphorylated RB1 appears as a distinct spot pattern compared to hyper-phosphorylated forms .

• Sequential immunoprecipitation and immunoblotting: First, immunoprecipitate RB1 using a phospho-specific antibody (e.g., anti-T821), then immunoblot with multiple phospho-specific antibodies targeting different sites. In mono-phosphorylated RB1, only the original phospho-site antibody will show positivity, while hyper-phosphorylated RB1 will be recognized by multiple phospho-specific antibodies .

• Cell cycle synchronization: Comparing samples from G0/early G1 phase (predominantly mono-phosphorylated RB1) with S phase samples (hyper-phosphorylated RB1) can help establish reference patterns .

Research by Narasimha et al. demonstrated that RB1 in early G1 phase is exclusively mono-phosphorylated, with 14 independent mono-phosphorylated isoforms (including T821) present . This finding explains the complex phosphorylation patterns previously observed in tryptic phospho-peptide mapping studies.

What are the optimal conditions for detecting phospho-T821 RB1 in stressed or apoptotic cells?

When studying phospho-T821 RB1 in stressed or apoptotic conditions, several methodological considerations are crucial:

• Timing of sample collection: T821 is rapidly dephosphorylated in response to cellular stress before other phospho-sites. Collect samples at multiple early time points (15, 30, 60 minutes) after stress induction to capture this dynamic change .

• Stress induction protocols:

  • Hypoxia: 1% O₂ atmosphere

  • DNA damage: Ara-C treatment

  • PNUTS knockdown: siRNA directed toward PNUTS to increase PP1 activity toward T821

• Phosphatase inhibitor considerations: Standard phosphatase inhibitor cocktails should be included in lysis buffers, but note that their use may mask the dephosphorylation of T821 that occurs during apoptotic signaling .

• Parallel assessment: Simultaneously measure T821 phosphorylation status alongside other phospho-sites (S780, S807/S811) to confirm the preferential dephosphorylation of T821 .

• Correlation with apoptotic markers: Co-stain for apoptotic markers (cleaved caspases, PARP cleavage) to establish temporal relationships between T821 dephosphorylation and apoptotic progression.

How can I validate the specificity of a Phospho-RB1 (T821) antibody in my experimental system?

Validating antibody specificity is critical for obtaining reliable results. For Phospho-RB1 (T821) antibodies, implement these validation strategies:

• Phosphopeptide competition assay: Pre-incubate the antibody with synthetic phosphopeptide containing the T821 phosphorylation site. This should abolish specific binding in your application of choice (WB, IHC, IF) .

• Dot blot analysis: Apply both phosphorylated and non-phosphorylated peptides to a nitrocellulose membrane and probe with the antibody. Specific antibodies will only detect the phosphorylated form .

• Cell cycle manipulation: Compare samples from cells in different cell cycle phases:

  • G0/G1 arrested cells (serum starvation): minimal T821 phosphorylation

  • S phase cells: maximal T821 phosphorylation

• Phosphatase treatment: Treat one set of samples with lambda phosphatase before immunoblotting to remove phosphate groups, which should eliminate signal from phospho-specific antibodies .

• Genetic controls: If possible, use RB1-null cell lines (e.g., certain retinoblastoma lines) as negative controls, or RB1 mutant constructs where T821 has been replaced with alanine (T821A) .

What are the optimal sample preparation methods for detecting phospho-T821 RB1 in different tissue types?

Sample preparation is critical for preserving phosphorylation status. Different tissue types require specific approaches:

For cell cultures:

• Harvest cells directly in denaturing lysis buffer containing phosphatase inhibitors to immediately prevent dephosphorylation.

• For adherent cells at 90% confluence, use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, with freshly added protease and phosphatase inhibitors .

• Process samples quickly on ice to prevent phosphatase activity.

For tissue samples:

• Flash-freeze harvested tissues in liquid nitrogen immediately after collection.

• For formalin-fixed paraffin-embedded (FFPE) tissues, perform antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) with microwave treatment for 8-15 minutes .

• Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 15 minutes at room temperature when performing IHC .

For both sample types:

• Optimize protein concentration for your specific application (typically 20-50 μg for Western blotting).

• Include positive controls (e.g., proliferating cancer cell lines) and negative controls (e.g., serum-starved cells) in every experiment.

How should I interpret conflicting phospho-T821 RB1 results across different experimental techniques?

When facing discrepancies between different experimental approaches measuring phospho-T821 RB1, consider these analytical strategies:

• Technical considerations first:

  • Antibody lot-to-lot variation: Different lots may have varying specificities or sensitivities

  • Sample preparation differences: Phosphorylation can be lost during lengthy procedures

  • Detection sensitivity thresholds: Some techniques (e.g., mass spectrometry) may be more sensitive than antibody-based methods

• Biological interpretations:

  • Cell cycle synchronization status: Heterogeneous cell populations will show mixed phosphorylation patterns

  • Mono-phosphorylation versus multiple phosphorylation: T821 may be phosphorylated alone or in combination with other sites

  • Dynamic phosphorylation/dephosphorylation: T821 phosphorylation can change rapidly with stress

• Resolution approach:

  • Implement multiple orthogonal techniques (WB, IF, IHC, mass spectrometry)

  • Compare data from synchronized cell populations

  • Use phospho-specific antibodies in combination with total RB1 antibodies to calculate phosphorylation ratios

  • Consider single-cell techniques to resolve population heterogeneity

What controls should be included when studying the functional significance of T821 phosphorylation?

To establish the functional significance of T821 phosphorylation in your research, incorporate these essential controls:

• Phospho-site mutants:

  • T821A (alanine substitution): Prevents phosphorylation at this site

  • T821E/D (glutamic acid/aspartic acid substitution): Phosphomimetic that simulates constitutive phosphorylation

• Additional phospho-site controls:

  • Single-site RB1 constructs with only T821 available for phosphorylation

  • Comparison with other phospho-sites (e.g., S807/S811, S780) to determine site-specific effects

• Enzymatic controls:

  • CDK inhibitors to prevent phosphorylation (e.g., palbociclib)

  • Phosphatase inhibitors to prevent dephosphorylation

  • PNUTS knockdown to increase PP1-mediated T821 dephosphorylation

• Functional readouts:

  • Cell cycle progression markers (cyclin E, PCNA)

  • E2F-dependent transcription assays

  • Apoptosis markers (caspase activation)

  • Specific gene expression changes (e.g., oxidative phosphorylation genes)

Studies have shown that mono-phosphorylation at T821 confers specific functional properties beyond simple cell cycle regulation, including impacts on oxidative phosphorylation gene expression and oxygen consumption .

How can phospho-T821 RB1 analysis be integrated into single-cell proteomic approaches?

Integrating phospho-T821 RB1 analysis into single-cell proteomics requires specialized approaches:

• Mass cytometry (CyTOF):

  • Conjugate phospho-T821 RB1 antibodies with rare earth metals

  • Combine with other phospho-specific antibodies and cell cycle markers

  • Allows correlation of T821 phosphorylation with cell cycle status at single-cell resolution

• Single-cell Western blotting:

  • Microfluidic platforms enable Western blotting on individual cells

  • Sequential probing with phospho-T821 and total RB1 antibodies provides phosphorylation ratio at single-cell level

  • Reveals heterogeneity masked in bulk population analyses

• Imaging mass spectrometry:

  • Allows spatial resolution of phospho-T821 RB1 within tissue sections

  • Can correlate with histopathological features in tumor samples

• Proximity ligation assays:

  • Detect interactions between phospho-T821 RB1 and binding partners

  • Provides spatial information within individual cells

These emerging technologies offer new insights into how T821 phosphorylation varies across individual cells in heterogeneous populations, with important implications for understanding tumor heterogeneity and therapeutic responses.

What are the potential research applications of phospho-T821 RB1 analysis in cancer therapeutics?

The site-specific phosphorylation of RB1 at T821 has several potential applications in cancer therapeutic research:

• CDK inhibitor response prediction:

  • The phosphorylation status of T821 may serve as a biomarker for sensitivity to CDK4/6 inhibitors

  • Monitoring T821 phosphorylation during treatment could provide early indicators of drug efficacy or resistance development

• Synthetic lethality screening:

  • Identify compounds that are selectively lethal to cells with aberrant T821 phosphorylation patterns

  • Develop combination therapies targeting cells with specific RB1 phosphorylation profiles

• Targeting RB1 phosphorylation-dependent functions:

  • Research shows that T821 mono-phosphorylation influences oxidative phosphorylation gene expression

  • This suggests potential for metabolic targeting in tumors with specific RB1 phosphorylation patterns

• Apoptosis sensitization strategies:

  • Since T821 dephosphorylation is associated with apoptotic responses , agents that promote specific dephosphorylation of this site might sensitize cancer cells to apoptotic stimuli

  • PNUTS inhibitors could potentially increase PP1 activity toward T821 and promote apoptosis in cancer cells

• Site-specific phosphorylation in different cancer types:

  • Creating a catalog of T821 phosphorylation patterns across cancer types

  • Correlating these patterns with clinical outcomes and therapeutic responses

How can computational approaches enhance our understanding of T821 phosphorylation in the context of the RB1 protein structure?

Computational methods offer powerful tools for understanding the structural and functional implications of T821 phosphorylation:

• Molecular dynamics simulations:

  • Model the conformational changes induced by T821 phosphorylation

  • Compare with other phosphorylation sites to identify unique structural consequences

  • Predict how these changes affect protein-protein interactions, particularly with E2F transcription factors

• Integrative structural biology:

  • Combine crystallographic data, NMR, and cryo-EM to build comprehensive models of RB1 in different phosphorylation states

  • Visualize how T821 phosphorylation alters the pocket domain and C-terminal interactions

• Protein interaction network analysis:

  • Predict potential phosphorylation-dependent interaction partners beyond E2F

  • Identify conditional protein interactions specific to T821 phosphorylation state

• Machine learning approaches:

  • Analyze proteomic data to identify patterns in phosphorylation combinations

  • Predict functional outcomes based on specific phosphorylation signatures

  • Develop predictive models for therapeutic responses based on phosphorylation patterns

• Systems biology modeling:

  • Integrate T821 phosphorylation into comprehensive cell cycle regulatory networks

  • Model the dynamic changes in T821 phosphorylation throughout the cell cycle and in response to various stimuli

  • Predict system-level consequences of altered T821 phosphorylation

These computational approaches can generate testable hypotheses about the structural basis for the unique functions of T821 phosphorylation observed in experimental studies.