Phospho-RB1 (S795) Antibody

What is the functional significance of RB1 phosphorylation at serine 795?

RB1 phosphorylation at serine 795 represents a critical regulatory mechanism in cell cycle control. This specific phosphorylation event directly inhibits RB1's association with E2F-DP heterodimers, thereby releasing E2F transcription factors from inhibition . The phosphorylation at S795 occurs primarily through cyclin-dependent kinase (CDK) activity, particularly CDK4/6-cyclin D complexes during the G1 phase, and serves as one of the key steps in inactivating RB1's tumor suppressor function . Research demonstrates that S795 phosphorylation is part of a coordinated phosphorylation program that regulates RB1's interaction with chromatin-modifying enzymes, ultimately influencing gene expression patterns related to cell proliferation .

How does mono-phosphorylation at S795 differ from hyper-phosphorylation of RB1?

Mono-phosphorylation at S795 represents a distinct regulatory state compared to hyper-phosphorylation:

Mono-phosphorylation at S795 creates a distinct functional state where RB1 selectively regulates subsets of genes, contrasting with hyper-phosphorylation which fully inactivates RB1 . Importantly, research has revealed that cells in early G1 phase contain exclusively mono-phosphorylated RB1, with no evidence of progressive hypo-phosphorylation as previously thought . Kinetic analyses have shown a quantum switch-like shift to hyper-phosphorylated RB1 coinciding with cyclin E:Cdk2 activation later in G1 phase .

How does RB1 S795 phosphorylation status change during normal cell cycle progression?

During cell cycle progression, S795 phosphorylation follows a defined temporal pattern:

• G0/quiescence: RB1 is predominantly unphosphorylated at S795

• Early G1 (upon mitogenic stimulation): Mono-phosphorylation at S795 appears within 1-3 hours

• Throughout early G1: RB1 remains exclusively mono-phosphorylated at S795 with active cyclin D:Cdk4/6 complexes

• Late G1/S transition: S795 becomes part of the hyper-phosphorylation pattern

• M phase: Dephosphorylation occurs during late M phase

Experimental data from both primary human fibroblasts (HFFs) and cancer cell lines (U2OS) confirm this pattern, demonstrating that S795 phosphorylation is tightly regulated across the cell cycle .

What are the optimal experimental conditions for using Phospho-RB1 (S795) antibodies in Western blot applications?

For optimal Western blot results with Phospho-RB1 (S795) antibodies:

Protocol Guidelines:

• Sample preparation: Use phosphatase inhibitors in lysis buffer to preserve phosphorylation status

• Protein loading: 20-50 μg of total protein per lane typically yields optimal signal

• Transfer conditions: Use PVDF membranes for optimal retention of phospho-epitopes

• Blocking: 5% BSA in TBST (not milk, which contains phosphatases)

• Antibody dilution: Recommended range of 1:500-1:1000

• Detection method: Enhanced chemiluminescence systems provide suitable sensitivity

Critical Considerations:

• Include positive control samples (e.g., cycling cells in late G1/S) and negative controls (e.g., serum-starved cells)

• For phospho-specificity validation, include lambda phosphatase-treated samples

• Expected molecular weight: 106-110 kDa for phosphorylated RB1

How can I optimize immunohistochemistry protocols for Phospho-RB1 (S795) antibody detection in tissue samples?

For successful IHC applications with Phospho-RB1 (S795) antibodies:

Protocol Optimization:

• Fixation: 10% neutral buffered formalin is recommended; prolonged fixation may mask phospho-epitopes

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) is generally effective

• Antibody concentration: Start with 1:50-1:200 dilution and optimize based on signal-to-noise ratio

• Detection system: Polymer-based detection systems provide enhanced sensitivity for phospho-epitopes

• Counterstaining: Hematoxylin provides good nuclear contrast while preserving phospho-RB1 signal

Validation Approaches:

• Use serial sections with total RB1 antibody to confirm expression pattern

• Include positive control tissues (e.g., proliferating tissues) and negative controls (e.g., G0-arrested tissues)

• Consider dual immunofluorescence with proliferation markers (Ki-67) for contextual interpretation

What are the key considerations when interpreting Phospho-RB1 (S795) data in cancer research applications?

When analyzing Phospho-RB1 (S795) data in cancer contexts:

Interpretative Framework:

• Cell cycle context: Interpret S795 phosphorylation in relation to cell cycle phase markers

• Heterogeneity assessment: Evaluate intratumoral variation in phosphorylation patterns

• Pathway integration: Analyze in context of CDK4/6 activity and upstream regulators (e.g., p16)

• Biological correlation: Connect phosphorylation status to proliferation indices and patient outcomes

Common Pitfalls:

• Misinterpreting mono-phosphorylation vs. hyper-phosphorylation states

• Failing to consider cell density effects (contact inhibition can cause selective dephosphorylation patterns)

• Not accounting for treatment effects (e.g., palbociclib treatment reduces but does not eliminate all RB1 phosphorylation forms)

How can Phospho-RB1 (S795) antibodies be used to study the differential effects of mono-phosphorylated RB1 isoforms?

Research into mono-phosphorylated RB1 isoforms represents an advanced application:

Experimental Approach:

• Quantitative proteomics: Use quantitative proteomics to profile protein complexes formed by S795 mono-phosphorylated RB1 compared to other mono-phosphorylated isoforms

• Transcriptional profiling: Couple with RNA-Seq to identify gene expression changes specific to S795 mono-phosphorylation versus other sites

• ChIP-seq analysis: Determine genomic binding patterns of S795 mono-phosphorylated RB1 versus other isoforms

• Phospho-mutant studies: Generate S795A (non-phosphorylatable) and S795D/E (phosphomimetic) mutants to dissect functional consequences

Research Insights:
Recent studies have shown that mono-phosphorylation at different sites creates "shades" of RB1 regulation, with S795 phosphorylation particularly affecting specific gene sets rather than global E2F regulation . For example, RB1 mono-phosphorylation at S795 alters its function by promoting distinct protein-protein interactions beyond canonical E2F regulation .

What methodologies can be used to study the impact of viral oncoproteins on RB1 S795 phosphorylation?

Given that viral oncoproteins specifically target RB1 function:

Experimental Design:

• Infection models: Establish cell models with viral oncoprotein expression (HPV E7, SV40 large T antigen, adenovirus E1A)

• Phosphorylation kinetics: Track S795 phosphorylation dynamics after viral oncoprotein expression using Western blot time-course analyses

• Protein-protein interactions: Perform co-IP assays with Phospho-RB1 (S795) antibodies to detect changes in interaction partners

• Subcellular localization: Use immunofluorescence with Phospho-RB1 (S795) antibodies to track localization changes induced by viral oncoproteins

Mechanistic Understanding:
Viral oncoproteins such as HPV E7 induce the disassembly of RB1-E2F1 complexes, disrupting RB1's tumor suppressor activity . S795 phosphorylation status serves as a marker for this disruption and may be mechanistically involved in the process, though the precise relationship between viral oncoprotein binding and site-specific phosphorylation patterns requires further investigation.

How do structural changes induced by S795 phosphorylation alter RB1's interaction with chromatin-modifying enzymes?

This represents an advanced research question addressing structural biology aspects:

Methodological Approaches:

• Structural analysis: Employ crystallography or cryo-EM to determine conformational changes induced by S795 phosphorylation

• Domain interaction studies: Investigate how S795 phosphorylation affects the interaction between RB1's C-terminal domain and pocket domain

• Chromatin-modifying enzyme binding assays: Use pull-down assays with specific phospho-states to identify differential binding of histone methyltransferases (e.g., SUV39H1, KMT5B, KMT5C)

• Histone modification profiling: Connect S795 phosphorylation status to changes in histone modifications, particularly H4K20 trimethylation

What are common sources of non-specific signal when using Phospho-RB1 (S795) antibodies and how can they be addressed?

Common Issues and Solutions:

Validation Approach:
Always validate phospho-specificity by treating parallel samples with lambda phosphatase. The disappearance of signal confirms phospho-specificity. Additionally, use RB1 knockout/knockdown cells as negative controls to confirm antibody specificity to the target protein.

How can I distinguish between specific Phospho-RB1 (S795) signaling and artifacts in complex tissue samples?

When working with tissues rather than cell lines:

Methodological Recommendations:

• Sequential Immunostaining: Perform sequential staining with total RB1 and phospho-specific antibodies on serial sections

• Dual Immunofluorescence: Co-stain with markers of proliferation (Ki-67) to contextualize S795 phosphorylation

• Spatial Analysis: Evaluate phospho-signal in relation to tissue architecture and cell cycle gradients

• Control Tissues: Include tissues known to be negative (e.g., terminally differentiated cells) and positive (e.g., proliferating germinal centers)

Advanced Validation:
For critical applications, consider phospho-peptide competition assays where excess synthesized phospho-peptide should abolish specific signal but not non-specific binding. This confirms the epitope specificity of the observed staining pattern.

How are Phospho-RB1 (S795) antibodies being used to study the relationship between RB1 phosphorylation and metabolic regulation?

Recent research has uncovered unexpected connections between RB1 phosphorylation and cellular metabolism:

Research Approaches:

• Metabolic profiling: Correlate S795 phosphorylation status with metabolomic data

• Mitochondrial function assays: Measure oxygen consumption and mitochondrial membrane potential in relation to S795 phosphorylation

• Gene expression analysis: Focus on oxidative phosphorylation genes regulated by mono-phosphorylated RB1

Emerging Findings:
Studies have revealed that specific mono-phosphorylation events, including at S795, can stimulate expression of oxidative phosphorylation genes and increase cellular oxygen consumption . This suggests RB1 phosphorylation states play roles beyond cell cycle regulation, potentially influencing metabolic adaptation in cancer cells.

What is the current understanding of how CDK inhibitors affect site-specific phosphorylation at S795 versus other RB1 phosphorylation sites?

With the clinical relevance of CDK4/6 inhibitors in cancer therapy:

Research Methodology:

• Phospho-site specific Western blotting: Compare phosphorylation at multiple sites (S795, S780, S807/S811) after CDK inhibitor treatment

• Temporal dynamics: Perform time-course analyses to identify site-specific dephosphorylation kinetics

• Cell type comparisons: Evaluate variation in S795 dephosphorylation patterns across cancer subtypes

Clinical Relevance:
Treatment with CDK4/6 inhibitors like palbociclib generally reduces RB1 phosphorylation, but not all phosphorylation isoforms are equally suppressed . This differential response may help explain variable therapeutic outcomes and resistance mechanisms. For example, in some cell lines, S795 phosphorylation may be maintained even when other sites are dephosphorylated, potentially preserving certain RB1 functions.