Phospho-RB1 (S811) Antibody

What is the biological significance of RB1 phosphorylation at Serine 811?

RB1 (Retinoblastoma protein) phosphorylation at Serine 811 plays a critical role in cell cycle regulation and broader cellular functions. Specifically:

• S811 phosphorylation is part of the CDK3/cyclin-C-mediated phosphorylation (along with S807) that is required for G0-G1 transition .

• Unlike hyper-phosphorylation which generally inhibits RB1's tumor suppressor properties, mono-phosphorylation at S811 maintains E2F binding while modulating its function .

• S811 phosphorylation alters RB1's transcriptional regulation by promoting its association with NuRD (Nucleosome Remodeling and Deacetylase) chromatin-remodeling complexes .

• Beyond cell cycle control, RB1 phosphorylated at S811 stimulates the expression of oxidative phosphorylation genes, significantly increasing cellular oxygen consumption .

This site-specific phosphorylation represents one component of what researchers have termed a "code of mono-phosphorylation" that determines the diverse activities of the RB1 protein in different cellular contexts.

How can I detect RB1 phosphorylation at S811 in my experimental system?

Several methodological approaches are available for detecting RB1 phosphorylation at S811:

Western Blot Analysis:

• Use phospho-specific antibodies targeting S811 with recommended dilutions of 1:1000-1:2000 .

• Include appropriate controls such as UV-treated HeLa cells, which show increased S811 phosphorylation .

• Consider dephosphorylation controls to validate antibody specificity.

Immunofluorescence/Immunohistochemistry:

• Multiple antibodies are validated for IF/IHC applications with recommended dilutions of 1:100-1:300 for IHC and 1:200-1:1000 for IF .

• Use appropriate fixation protocols to preserve phospho-epitopes.

HTRF (Homogeneous Time-Resolved Fluorescence) Assay:

• For quantitative detection, HTRF cell-based assays offer a no-wash format that accurately quantifies phosphorylated RB at Ser807/811 .

• This plate-based method uses two labeled antibodies (donor and acceptor fluorophores) to generate a FRET signal proportional to phospho-RB concentration .

ELISA:

• Multiple antibodies are validated for ELISA applications with product-specific recommended dilutions .

• Follow manufacturer protocols for optimal results.

What is the relationship between S811 phosphorylation and cell cycle progression?

The relationship between S811 phosphorylation and cell cycle progression is complex and context-dependent:

• CDK3/cyclin-C-mediated phosphorylation at S807 and S811 is specifically required for the G0-G1 transition, distinct from other phosphorylation events during later cell cycle phases .

• In contact-inhibited cells (RPE1, CAMA1), phosphorylation at S807/S811 decreases, while in T47D cells treated with gamma-irradiation or hydroxyurea, S807/S811 phosphorylation is selectively maintained .

• Unlike other phosphorylation sites that completely inactivate RB1, mono-phosphorylation at S811 creates a functionally active RB1 that still arrests cells in G1-phase while modulating specific gene expression programs .

This site-specific phosphorylation illustrates how different phosphorylation events can fine-tune RB1 function rather than simply activating or inactivating the protein, representing a sophisticated regulatory mechanism.

How should I optimize Western Blot protocols for reliable Phospho-RB1 (S811) detection?

For optimal Western Blot detection of Phospho-RB1 (S811):

Sample Preparation:

• Rapid cell lysis is critical to preserve phosphorylation states - use phosphatase inhibitors in all buffers.

• For UV-induced RB1 phosphorylation protocols, treat HeLa cells with UV for 15-30 minutes prior to lysis .

• Use 25μg protein per lane for reliable detection .

Blocking and Antibody Incubation:

• Use 3% BSA rather than milk for blocking, as milk contains phosphatases that may reduce signal .

• Secondary antibody recommendations: HRP-conjugated anti-Rabbit IgG at 1:10000 dilution .

Controls and Validation:

• Include both phosphorylated (e.g., UV-treated) and non-phosphorylated samples.

• Consider lambda phosphatase treatment of duplicate samples to confirm specificity.

• Be aware that different cell treatments may produce different RB1 phosphoisoforms - CDK4/6 inhibitors like palbociclib generally reduce RB phosphorylation, but not all phosphorylation sites are equally suppressed .

Detection and Analysis:

• RB1 phospho-S811 runs at approximately 106 kDa molecular weight .

• When analyzing results, consider that phosphorylation patterns vary by cell type and treatment, reflecting cell-specific regulatory mechanisms .

How can I distinguish between the functions of mono-phosphorylation at S811 versus other phosphorylation sites?

Distinguishing the specific functions of S811 phosphorylation requires sophisticated experimental approaches:

Site-Specific Mutants:

Proteomic Analysis:

• Quantitative proteomics can profile protein complexes associated with different mono-phosphorylated RB1 isoforms.

• This approach revealed that S811 phosphorylation promotes association with NuRD complexes, while other sites form different protein interactions .

Transcriptional Profiling:

• RNA-seq analysis comparing cells expressing different mono-phosphorylated RB1 isoforms can identify site-specific transcriptional outputs.

• This approach demonstrated that S811 phosphorylation specifically enhances oxidative phosphorylation gene expression .

Metabolic Analysis:

• For oxidative phosphorylation effects, oxygen consumption measurements can be used to validate functional consequences of S811 phosphorylation.

• Cells with RB1 phosphorylated at S811 or T826 showed elevated cellular oxygen consumption compared to other phosphorylation sites .

These methodologies allow researchers to move beyond simple detection of phosphorylation and into understanding the functional significance of site-specific modifications.

What controls should I implement when studying RB1 S811 phosphorylation in cancer models?

When studying RB1 S811 phosphorylation in cancer models, implement these critical controls:

RB1 Status Verification:

• Confirm RB1 expression status in your cancer model, as many tumor lines have lost RB1 expression.

• Verify total RB1 protein levels alongside phosphorylation status to normalize phospho-signals appropriately.

Cell Cycle Synchronization:

• Since phosphorylation varies throughout the cell cycle, synchronize cells or use cell cycle markers to properly interpret phosphorylation patterns.

• Different cancer cell lines show distinct phosphorylation responses to the same treatments - for example, gamma-irradiation generates different RB1 phosphorylation patterns in T47D versus MDA-MB-361 cells .

Drug Treatment Controls:

• Include CDK4/6 inhibitor controls (e.g., palbociclib) to determine which phosphorylation events are CDK4/6-dependent .

• Different treatments may preferentially affect specific phosphorylation sites - hydroxyurea in T47D cells selectively maintains S807/S811 phosphorylation, while camptothecin in MDA-MB-361 preferentially reduces S780 and S795 phosphorylation .

Viral Oncogene Considerations:

• If studying viral-associated cancers, consider that viral oncoproteins (SV40 large T antigen, HPV E7, adenovirus E1A) interact with RB1 and disrupt its function through mechanisms that may mask or alter phosphorylation patterns .

These controls ensure proper interpretation of phosphorylation data in the context of the complex regulatory network affecting RB1 in cancer cells.

How can I design experiments to specifically study S811 phosphorylation's role in oxidative phosphorylation?

To investigate the specific role of S811 phosphorylation in regulating oxidative phosphorylation:

Cellular Models:

• Utilize the established isogenic cell system with RB1 mutants that can only be phosphorylated at single sites .

• Compare S811-phosphorylated RB1 with other phospho-sites such as T826 (which also affects oxidative phosphorylation) and phospho-sites that do not impact this pathway.

Gene Expression Analysis:

• Conduct RNA-seq or targeted qPCR analysis of nuclear-encoded mitochondrial genes in cells expressing different RB1 phospho-mutants.

• Focus on oxidative phosphorylation genes that are specifically upregulated by S811 phosphorylation .

Metabolic Assays:

• Measure oxygen consumption rates using platforms like Seahorse XF Analyzer to quantify functional changes in mitochondrial respiration.

• Assess ATP production, mitochondrial membrane potential, and ROS generation as additional functional readouts.

Mechanistic Investigations:

• Analyze NuRD complex recruitment to metabolic gene promoters using ChIP-seq, as S811 phosphorylation promotes association with NuRD complexes .

• Investigate potential direct binding of S811-phosphorylated RB1 to regulatory regions of metabolic genes.

In vivo Validation:

• If possible, generate knock-in mouse models with S811 phospho-mimetic or non-phosphorylatable mutations.

• Assess tissue-specific metabolic consequences, particularly in tissues with high oxidative metabolism.

This experimental approach separates S811's metabolic functions from its cell cycle regulatory roles, providing insight into this unique aspect of RB1 biology.

What kinases are responsible for S811 phosphorylation and how can they be manipulated experimentally?

Several kinases are implicated in RB1 S811 phosphorylation, providing multiple experimental manipulation strategies:

CDK/Cyclin Complexes:

• CDK3/cyclin-C is specifically implicated in phosphorylating S807 and S811 during G0-G1 transition .

• CDK4/6 inhibitors like palbociclib generally reduce RB1 phosphorylation, though the effect varies by site and cell type .

• Experimental approaches include:

  • Small molecule inhibitors specific to different CDKs

  • siRNA/shRNA knockdown of specific CDKs or cyclins

  • Expression of dominant-negative CDK mutants

Non-CDK Kinases:

• p38 MAPK has been implicated in phosphorylating RB1 under stress conditions, though specific sites have not been fully characterized .

• Under DNA damage conditions, RB1 phosphorylation patterns differ from normal S-phase patterns, suggesting involvement of other kinases .

• Experimental approaches include:

  • Specific p38 MAPK inhibitors (SB203580, BIRB796)

  • Activation of p38 pathways with anisomycin or UV treatment

  • Stress-inducing agents that activate distinct kinase pathways

Phosphatase Regulation:

• Calcineurin-mediated dephosphorylation affects RB1 phosphorylation state .

• Experimental approaches include:

  • Calcineurin inhibitors (cyclosporin A, FK506)

  • Calcium ionophores to activate calcineurin

  • Phosphatase inhibitors (okadaic acid, calyculin A) to broadly preserve phosphorylation

When manipulating these enzymes, careful consideration of cell cycle position and broader signaling effects is crucial for proper interpretation of S811-specific effects.

How should I interpret variations in S811 phosphorylation patterns across different cellular contexts?

Interpreting variations in RB1 S811 phosphorylation requires consideration of multiple contextual factors:

Cell Type Heterogeneity:

• Different cell lines show distinct patterns of RB1 phosphorylation even under similar conditions.

• For example, contact inhibition causes dephosphorylation of multiple sites including S807/S811 in RPE1 and CAMA1 cells, while gamma-irradiation in T47D cells selectively maintains S807/S811 phosphorylation .

• Interpretation should consider the cell's tissue of origin, transformation status, and endogenous signaling pathways.

Treatment-Specific Responses:

• The same cell line can show different phosphorylation patterns depending on treatment:

  • Palbociclib (CDK4/6 inhibitor) reduces most RB1 phosphorylation sites

  • Camptothecin preferentially reduces S780 and S795 phosphorylation in MDA-MB-361 cells

  • Hydroxyurea maintains S807/S811 phosphorylation in T47D cells

• These differential responses may reflect treatment-specific activation of kinases or phosphatases.

Cell Cycle Confounding:

• While cell cycle position influences phosphorylation, the research shows that changes in RB1 phosphorylation cannot simply be attributed to differences in cell cycle position .

• This suggests additional regulatory mechanisms beyond standard cell cycle-coupled phosphorylation events.

Functional Interpretation:

• Mono-phosphorylation at S811 maintains RB1's ability to arrest cells in G1 while also promoting specific functions like NuRD complex association and oxidative phosphorylation gene expression .

• Therefore, detecting S811 phosphorylation should not simply be interpreted as RB1 inactivation, but rather as functional modulation.

This context-dependent interpretation reflects the emerging understanding of RB1 phosphorylation as a code rather than a simple on/off switch.

How does S811 phosphorylation data integrate with broader RB1 functional studies?

Integrating S811 phosphorylation data with broader RB1 studies requires a multifaceted approach:

Relationship to RB1's Tumor Suppressor Function:

• While hyper-phosphorylation generally inactivates RB1's tumor suppressor function, mono-phosphorylation at S811 maintains G1 arrest capability while modulating specific functions .

• This challenges the simplified view of phosphorylation as merely inactivating RB1.

Chromatin Regulation Context:

• RB1 is directly involved in heterochromatin formation and histone modification .

• S811 phosphorylation promotes association with NuRD chromatin-remodeling complexes .

• Consider analyzing histone modifications (especially H4K20 trimethylation) and chromatin accessibility near RB1 target genes when studying S811 phosphorylation.

Mitochondrial Function Connection:

• The strong link between S811 phosphorylation and oxidative phosphorylation gene expression provides a unique connection to metabolic regulation .

• This represents a pathway-specific effect distinct from general cell cycle control.

• Consider metabolic parameters alongside traditional RB1 readouts when studying S811 phosphorylation.

E2F Interaction Nuances:

• All mono-phosphorylated RB1 forms, including S811, interact with E2F/DP proteins, but they provide "different shades of E2F regulation" .

• This suggests that S811 phosphorylation creates a functionally distinct RB1-E2F interaction rather than simply disrupting it.

• Analyzing E2F target gene expression patterns can help distinguish S811-specific effects from other phosphorylation events.

By contextualizing S811 phosphorylation within these broader aspects of RB1 biology, researchers can develop more sophisticated models of RB1 regulation and function.

What considerations should be made when comparing Phospho-RB1 (S811) data across different experimental methodologies?

When comparing Phospho-RB1 (S811) data across different methodologies, consider these technical factors:

Antibody Specificity Variations:

• Different antibodies may have varying specificity for RB1 phosphorylated only at S811 versus S807+S811 dual phosphorylation.

• Some antibodies specifically detect dual phosphorylation at S807+S811 , while others target S811 alone .

• Cross-reactivity with other phosphorylation sites may confound interpretation.

Detection Method Sensitivity:

• Western blot provides information about total molecular weight and can detect multiple phosphorylation states.

• HTRF and ELISA provide quantitative measurements but may not distinguish mono- from multi-phosphorylated forms.

• Immunofluorescence provides spatial information but may lack quantitative precision.

Sample Preparation Impacts:

• Phosphorylation states can be rapidly lost during sample preparation without adequate phosphatase inhibition.

• Different lysis buffers may preferentially extract different subcellular pools of RB1.

• Fixation methods for immunofluorescence/IHC can affect phospho-epitope accessibility.

Standardization Approaches:

• When comparing across methods, include common positive controls (e.g., UV-treated HeLa cells) .

• Use phospho-specific and total RB1 antibodies to normalize for protein expression differences.

• Consider using phosphatase-treated samples as negative controls.

Quantification Methodologies:

• Western blot densitometry measurements should be normalized to total RB1.

• HTRF signal should be reported as the 665nm/620nm emission ratio .

• Immunofluorescence quantification should account for nuclear versus cytoplasmic signals.

These considerations ensure that observed differences reflect true biological variation rather than methodological artifacts, allowing for more reliable integration of data across different experimental platforms.

How can Phospho-RB1 (S811) detection be utilized in cancer research?

Phospho-RB1 (S811) detection offers several valuable applications in cancer research:

CDK4/6 Inhibitor Response Prediction:

• As CDK4/6 inhibitors like palbociclib affect RB1 phosphorylation, monitoring S811 phosphorylation may help predict or assess treatment response .

• Different cancer cell lines show distinct patterns of RB1 phosphorylation in response to the same treatments, potentially informing personalized therapeutic approaches.

Cell Cycle Checkpoint Analysis:

• S811 phosphorylation's role in G0-G1 transition makes it a valuable marker for studying cell cycle checkpoint dysregulation in cancer .

• Unlike other sites, S811 phosphorylation may contribute to a functionally active RB1 while still permitting specific transcriptional programs.

Metabolic Reprogramming Studies:

• The unique connection between S811 phosphorylation and oxidative phosphorylation gene expression relates directly to the Warburg effect and metabolic reprogramming in cancer .

• Monitoring S811 phosphorylation alongside metabolic parameters could reveal novel aspects of tumor metabolism regulation.

DNA Damage Response Investigation:

• In T47D cells, gamma-irradiation or hydroxyurea treatment generates RB1 selectively phosphorylated on S807/S811, suggesting a role in DNA damage response .

• This could inform studies on how cancer cells adapt to genotoxic therapies.

Viral Oncogenesis Models:

• Interactions between viral oncoproteins (SV40 large T antigen, HPV E7, adenovirus E1A) and RB1 may affect S811 phosphorylation patterns .

• This could provide insights into mechanisms of viral carcinogenesis.

These applications highlight how site-specific phosphorylation analysis can advance beyond simple RB1 status assessment to more sophisticated understanding of cancer biology.

What are emerging research questions regarding the mono-phosphorylation code of RB1?

Several frontier research questions are emerging regarding the RB1 mono-phosphorylation code:

Combinatorial Phosphorylation Patterns:

• How do different combinations of mono-phosphorylation sites create distinct functional outcomes?

• Is there a hierarchy or sequence to phosphorylation events at different sites?

• Do certain mono-phosphorylation events prime RB1 for subsequent modifications?

Tissue-Specific Regulation:

• Do different tissues exhibit characteristic RB1 mono-phosphorylation patterns?

• How do tissue-specific kinases and phosphatases contribute to unique phosphorylation codes?

• Are developmental transitions associated with specific changes in mono-phosphorylation patterns?

Non-Canonical Functions:

• Beyond oxidative phosphorylation, what other non-canonical pathways are regulated by specific mono-phosphorylation events?

• How does mono-phosphorylation at sites like S811 contribute to RB1's roles in differentiation, senescence, and apoptosis?

Structural Consequences:

• How do different mono-phosphorylation events alter RB1 protein structure?

• While phosphorylation at T821 and T826 promotes intramolecular interactions, and T373 promotes conformational changes, what structural changes result from S811 phosphorylation?

Therapeutic Targeting:

• Can specific kinases responsible for S811 phosphorylation be targeted for therapeutic benefit?

• Would selective inhibition of certain phosphorylation sites provide therapeutic advantages over broad CDK inhibition?

• Could metabolic therapies be effectively combined with approaches that modulate S811 phosphorylation?

These questions represent emerging directions in understanding how the complex phosphorylation code of RB1 contributes to its diverse biological functions.

What methodological advances would improve S811 phosphorylation research?

Several methodological advances would significantly advance Phospho-RB1 (S811) research:

Advanced Antibody Development:

• Generation of antibodies with higher specificity for mono-phosphorylated S811 versus dual-phosphorylated S807/S811.

• Development of conformation-specific antibodies that recognize distinct structural states induced by S811 phosphorylation.

• Creation of antibodies compatible with ChIP-seq to map genomic binding sites of S811-phosphorylated RB1.

Mass Spectrometry Approaches:

• Improved enrichment techniques for phosphopeptides containing S811.

• Targeted mass spectrometry assays to quantify S811 phosphorylation in complex samples.

• Development of methods to quantify multiple RB1 phosphorylation sites simultaneously to capture combinatorial patterns.

Cellular Models:

• CRISPR knock-in models with endogenous RB1 mutations that prevent or mimic S811 phosphorylation.

• Development of optogenetic tools to rapidly and reversibly induce or inhibit S811 phosphorylation.

• Patient-derived models to study S811 phosphorylation in the context of different genetic backgrounds.

In vivo Techniques:

• Development of mouse models with specific S811 mutations.

• Tissue-specific expression of phospho-site mutants to evaluate organ-specific functions.

• Development of imaging techniques to visualize S811 phosphorylation dynamics in living tissues.

Computational Approaches:

• Machine learning algorithms to predict functional consequences of S811 phosphorylation in different cellular contexts.

• Network analysis tools to integrate S811 phosphorylation data with broader signaling pathways.

• Molecular dynamics simulations to predict structural changes induced by S811 phosphorylation.