Phospho-RB1 (S608) Antibody

What is the biological significance of RB1 phosphorylation at serine 608?

Phosphorylation of RB1 at serine 608 is a key regulatory event in the cell cycle. The Retinoblastoma protein functions as a tumor suppressor and negative regulator of the cell cycle. When in its hypophosphorylated state, RB1 binds to E2F family transcription factors, preventing the transcription of E2F-responsive genes necessary for cell cycle progression. Phosphorylation at S608, which depends on CDK4, contributes to the inactivation of RB1's growth-suppressive function, allowing E2F release and subsequent entry into S phase . This phosphorylation event is particularly important in understanding the mechanisms of cancer development and progression, as dysregulation of this process can lead to uncontrolled cell proliferation .

How does S608 phosphorylation differ functionally from phosphorylation at other RB1 sites?

While RB1 contains multiple phosphorylation sites, S608 phosphorylation has distinct functional implications. Unlike phosphorylation at S807/811 which has been more strongly associated with RB1's role in apoptosis and interaction with Bax , S608 phosphorylation appears more specifically involved in cell cycle regulation through E2F binding. Research indicates that various phosphorylation sites work in concert, with S608 being part of a coordinated phosphorylation program. Experimental approaches comparing the effects of phosphorylation-site mutants have revealed that S608 phosphorylation contributes to conformational changes that affect pocket domain interactions with E2F transcription factors .

What are the optimal protocols for using Phospho-RB1 (S608) antibodies in Western blotting?

For optimal Western blotting results with Phospho-RB1 (S608) antibodies:

• Sample preparation: Use fresh cell or tissue lysates extracted with phosphatase inhibitors to preserve phosphorylation status.

• Dilution ratios: Most Phospho-RB1 (S608) antibodies work optimally at dilutions between 1:500-1:2000 .

• Observed molecular weight: Expect to detect bands at approximately 110 kDa .

• Positive controls: Jurkat cell lysates are recommended as positive controls .

• Blocking: Use 5% BSA in TBST rather than milk (which contains phosphatases).

• Detection system: An enhanced chemiluminescence system provides sensitive detection of phosphorylated epitopes.

For recombinant monoclonal antibodies like EPR10849, a dilution of 1:1000 is typically recommended , while polyclonal antibodies may require optimization within the 1:500-1:2000 range .

What are the technical considerations for immunohistochemistry applications with Phospho-RB1 (S608) antibodies?

When performing IHC with Phospho-RB1 (S608) antibodies:

• Fixation: Formalin-fixed, paraffin-embedded (FFPE) tissues are commonly used.

• Antigen retrieval: Most protocols require heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Recommended dilutions: For IHC applications, dilutions typically range from 1:100-1:300 .

• Detection systems: Both manual and automated immunostaining systems can be used, as demonstrated in studies utilizing Leica Bond Refine polymer detection systems .

• Controls: Include phosphatase-treated sections as negative controls to verify phospho-specificity.

• Subcellular localization: Expect predominantly nuclear staining, consistent with RB1's cellular localization .

How can researchers validate the specificity of Phospho-RB1 (S608) antibodies in their experimental systems?

To validate antibody specificity:

• Phosphatase treatment: Treat half of your sample with lambda phosphatase to remove phosphorylation, which should eliminate signal from a truly phospho-specific antibody.

• Knockout/knockdown controls: Use RB1-null cell lines or RB1-knockdown samples as negative controls.

• Peptide competition assays: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides; only the phosphorylated peptide should block specific binding.

• Immunoprecipitation validation: Perform IP followed by Western blot with another RB1 antibody.

• Compare multiple phospho-specific antibodies: Use antibodies from different vendors that recognize the same phosphorylation site.

• Induction experiments: Compare samples treated with agents known to induce or reduce S608 phosphorylation (e.g., CDK4/6 inhibitors should reduce signal).

What are the relative advantages of monoclonal versus polyclonal Phospho-RB1 (S608) antibodies?

Selection should be based on the specific experimental requirements, with monoclonals like EPR10849 offering high reproducibility for mechanistic studies , while polyclonals might provide advantages in detection sensitivity .

How do different phospho-specific RB1 antibodies (S608, S807/811, S795) compare in research applications?

Different phospho-specific RB1 antibodies reveal distinct aspects of RB1 function:

• S608 phosphorylation:

  • Primary role in cell cycle regulation and E2F interaction

  • Dependent on CDK4 activity

  • Commonly used in cell cycle studies and cancer research

  • Detected at 110 kDa by Western blot in human samples

• S807/811 phosphorylation:

  • Stronger association with RB1's role in apoptosis

  • More readily detected in Bax interaction studies

  • Required for G0-G1 transition

  • Mediated by CDK3/cyclin-C

• S795 phosphorylation:

  • Less strongly associated with Bax interaction than S807/811

  • Can be dephosphorylated by calcineurin upon calcium stimulation

  • Important in cell cycle control mechanisms

Experimental evidence shows that while all these phosphorylation events can be detected in immunoprecipitation studies with Bax, the S807/811 phosphorylation shows the strongest signal, followed by S608 and S795, which require longer exposure times for detection .

How can Phospho-RB1 (S608) antibodies be used to investigate cancer-specific alterations in the RB pathway?

Phospho-RB1 (S608) antibodies provide valuable tools for investigating cancer-specific RB pathway alterations:

• Alternative mechanism detection: Studies have used phospho-S608 antibodies to identify tumors that inactivate the RB pathway through hyperphosphorylation rather than genetic mutation of RB1 .

• Multi-parameter analysis: Combine phospho-S608 detection with other markers:

  • CDK4/6 expression levels

  • Cyclin D1 overexpression

  • p16INK4a status

  • E2F target gene expression

• Tissue microarray analysis: Screen multiple tumor types to identify patterns of RB1 phosphorylation across cancer subtypes. This has been particularly informative in studies of breast, colon, prostate, kidney, and nasopharyngeal cancers .

• Treatment response monitoring: Monitor changes in S608 phosphorylation following treatment with CDK inhibitors or other targeted therapies.

• Correlation with clinical outcomes: Assess whether S608 phosphorylation status correlates with patient prognosis or treatment response.

Research has shown that phosphorylation of pRb at S608 can be detected in tumors without coding alterations in RB1, suggesting this as an alternative mechanism of RB pathway inactivation in cancer development .

What experimental designs are optimal for studying the relationship between CDK4 activity and RB1 S608 phosphorylation?

To study the relationship between CDK4 activity and RB1 S608 phosphorylation:

• CDK4 inhibition studies:

  • Treat cells with selective CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib)

  • Monitor S608 phosphorylation over time using Western blot

  • Compare with other phosphorylation sites to determine specificity

• Genetic manipulation:

  • Express wild-type, dominant-negative, or constitutively active CDK4 constructs

  • Generate CDK4 knockout or knockdown cell lines using CRISPR-Cas9 or RNAi

  • Assess changes in S608 phosphorylation status

• Cell synchronization experiments:

  • Synchronize cells at different cell cycle phases

  • Analyze correlation between CDK4 activity and S608 phosphorylation throughout the cell cycle

  • Use double-labeling with cell cycle markers

• In vitro kinase assays:

  • Purify CDK4/cyclin D complexes

  • Perform in vitro kinase reactions with recombinant RB1 protein or peptides

  • Use phospho-specific antibodies to detect site-specific phosphorylation

• Phosphoproteomic analysis:

  • Perform mass spectrometry to quantitatively assess changes in multiple RB1 phosphorylation sites

  • Compare phosphorylation patterns after CDK4 manipulation

Research has established that phosphorylation of RB1 at S608 specifically depends on CDK4 activity, distinguishing it from other phosphorylation events mediated by different CDKs .

How do post-translational modifications interact with S608 phosphorylation to regulate RB1 function?

RB1 function is regulated by a complex interplay of post-translational modifications (PTMs):

• Phosphorylation crosstalk:

  • S608 phosphorylation occurs in coordination with other phosphorylation events

  • The sequential phosphorylation model suggests certain sites must be phosphorylated before others

  • Evidence indicates CDK3/cyclin-C-mediated phosphorylation at S807/S811 is required for G0-G1 transition, while S608 phosphorylation by CDK4 regulates G1/S transition

• Methylation-phosphorylation interaction:

  • Monomethylation at Lys-810 by SMYD2 enhances phosphorylation at Ser-807 and Ser-811

  • N-terminal methylation by METTL11A/NTM1 may influence phosphorylation patterns

  • Monomethylation at Lys-860 by SMYD2 promotes interaction with L3MBTL1

• Acetylation effects:

  • RB1 undergoes acetylation during keratinocyte differentiation

  • Acetylation at Lys-873 and Lys-874 occurs during cellular stress and DNA damage

  • These events may modulate the effects of phosphorylation

• Experimental approaches:

  • Use site-specific mutants (phosphomimetic or phospho-deficient)

  • Apply specific kinase or acetyltransferase inhibitors

  • Employ phosphatase treatments alongside other PTM-specific enzymes

  • Perform sequential ChIP experiments to determine occupancy of differently modified RB1

Research has shown that these diverse modifications create a complex "RB1 code" that determines its binding partners and functional outcomes in different cellular contexts .

What are common issues when using Phospho-RB1 (S608) antibodies and how can they be resolved?

For optimal specificity in detecting endogenous phosphorylation, consider using rabbit-derived antibodies which demonstrate excellent reactivity with human, rat, and monkey samples when used at recommended dilutions .

How should researchers design control experiments to validate Phospho-RB1 (S608) antibody results?

Comprehensive validation controls include:

• Phosphatase controls:

  • Treat duplicate samples with lambda phosphatase

  • Compare phosphatase-treated vs. untreated samples by Western blot

  • Signal should disappear in phosphatase-treated samples

• Genetic controls:

  • Use RB1-null cell lines (e.g., certain SCLC lines)

  • Compare with RB1-expressing cell lines

  • No signal should be detected in RB1-null cells

• Stimulation/inhibition controls:

  • Serum-starve cells (reduces phosphorylation)

  • Treat with CDK4/6 inhibitors (reduces S608 phosphorylation)

  • Stimulate with growth factors (increases phosphorylation)

• Peptide competition:

  • Pre-incubate antibody with phosphorylated peptide

  • Pre-incubate with non-phosphorylated peptide

  • Only phospho-peptide should block specific signal

• Multiple antibody validation:

  • Compare results between different antibody clones/vendors

  • Use both monoclonal and polyclonal antibodies if possible

• Positive control samples:

  • Include Jurkat cell lysates as recommended positive controls

  • Use cell lines known to have hyperphosphorylated RB1

These controls ensure that observed signals truly represent S608 phosphorylation rather than artifacts or non-specific binding.

How is Phospho-RB1 (S608) detection being incorporated into studies of chromatin modification and gene regulation?

Emerging research applications include:

These applications are revealing how dynamic phosphorylation of RB1 at S608 contributes to epigenetic regulation across different cellular contexts.

What is the significance of RB1 S608 phosphorylation in therapeutic response to CDK4/6 inhibitors?

The phosphorylation status of RB1 at S608 has significant implications for CDK4/6 inhibitor therapy:

• Predictive biomarker potential:

  • S608 phosphorylation levels may predict sensitivity to CDK4/6 inhibitors

  • Antibody-based detection can be used to stratify patients for clinical trials

  • Monitoring changes in phosphorylation during treatment may provide early indicators of response

• Resistance mechanism identification:

  • Persistent S608 phosphorylation despite CDK4/6 inhibition suggests alternative kinases are active

  • This pattern may identify tumors developing resistance to CDK4/6 inhibitors

• Combination therapy rationale:

  • Understanding the relationship between S608 phosphorylation and other signaling pathways can guide rational combination therapies

  • Targeting multiple pathways affecting RB1 phosphorylation may overcome resistance

• Experimental approaches:

  • Time-course analysis of S608 phosphorylation after CDK4/6 inhibitor treatment

  • Correlation of phosphorylation status with clinical response

  • Comparison with other phosphorylation sites to determine site-specific effects

• Translation to clinical practice:

  • Development of IHC-based assays for clinical samples

  • Standardization of S608 phosphorylation assessment for patient selection

Research on S608 phosphorylation provides mechanistic insights into how CDK4/6 inhibitors exert their effects and may help identify patients most likely to benefit from these targeted therapies.

What criteria should researchers use when selecting between different commercial Phospho-RB1 (S608) antibodies?

When selecting Phospho-RB1 (S608) antibodies, consider:

• Application-specific validation:

  • Verify the antibody has been validated for your specific application (WB, IHC, IF, ELISA)

  • Review published literature using the antibody for similar applications

  • Examine validation data showing specificity in relevant experimental contexts

• Host species and format:

  • Rabbit-derived antibodies show good reactivity across human, rat, and monkey samples

  • Consider format (unconjugated vs. conjugated) based on detection method

  • Mouse monoclonals may be advantageous for co-labeling with rabbit antibodies

• Clonality considerations:

  • Monoclonal antibodies (e.g., EPR10849, 51B7) offer high reproducibility

  • Polyclonal antibodies may provide higher sensitivity for low abundance targets

• Epitope specificity:

  • Review immunogen information

  • Check sequence alignment with other RB1 phosphorylation sites

  • Consider antibodies raised against longer vs. shorter peptide sequences

• Reactivity with species of interest:

  • Verify cross-reactivity with your experimental system

  • Note that some antibodies are specifically validated for human samples only

  • Others work across multiple species including human, mouse, and rat

• Technical support and validation data:

  • Evaluate the quality and comprehensiveness of technical documentation

  • Look for actual experimental images rather than just statements of reactivity

  • Consider the manufacturer's reputation for antibody validation

Selecting the appropriate antibody based on these criteria ensures more reliable and reproducible experimental results.

How can phospho-specific flow cytometry be optimized for measuring RB1 S608 phosphorylation in heterogeneous cell populations?

Optimizing phospho-flow for RB1 S608 analysis:

• Cell fixation and permeabilization:

  • Test multiple fixation protocols (paraformaldehyde, methanol)

  • Compare permeabilization agents (saponin, Triton X-100)

  • Optimize timing to preserve phospho-epitopes while allowing antibody access

• Antibody selection and validation:

  • Test both monoclonal and polyclonal antibodies for flow performance

  • Verify specificity with phosphatase-treated controls

  • Titrate antibody concentration for optimal signal-to-noise ratio

• Multi-parameter analysis:

  • Include cell cycle markers (DNA content, Ki-67)

  • Add surface markers to identify specific cell subpopulations

  • Consider multiplexing with other phospho-proteins (pCDK4, other pRB sites)

• Controls and standardization:

  • Use phosphatase-treated cells as negative controls

  • Include cells with known high S608 phosphorylation as positive controls

  • Implement fluorescence minus one (FMO) controls for accurate gating

• Data analysis considerations:

  • Analyze phosphorylation relative to cell cycle phase

  • Use appropriate statistical methods for heterogeneous populations

  • Consider phosphorylation index relative to total RB1 expression

This approach enables quantitative assessment of S608 phosphorylation across different cell subpopulations, providing insights into heterogeneity of RB pathway activation within complex samples.

What are promising new applications for Phospho-RB1 (S608) antibodies in cancer research and personalized medicine?

Emerging applications include:

• Liquid biopsy development:

  • Detection of phosphorylated RB1 in circulating tumor cells

  • Correlation with disease progression and treatment response

  • Non-invasive monitoring of RB pathway activation

• Single-cell analysis:

  • Integration with single-cell proteomics

  • Mapping heterogeneity of RB1 phosphorylation in tumors

  • Identifying rare cell populations with altered phosphorylation

• Therapeutic response prediction:

  • Development of companion diagnostics for CDK4/6 inhibitors

  • Standardized IHC assays for clinical decision-making

  • Integration into multi-biomarker predictive panels

• Targeted protein degradation:

  • Monitoring phosphorylation-dependent degradation of RB1

  • Assessing effects of novel targeted protein degradation therapeutics

  • Studying phosphorylation-dependent protein-protein interactions

• Spatial biology:

  • Implementation in multiplexed tissue imaging platforms

  • Analysis of spatial relationships between phosphorylated RB1 and other markers

  • Tumor microenvironment effects on RB1 phosphorylation

These forward-looking applications could transform how we understand and target the RB pathway in cancer, moving beyond traditional research applications toward clinically actionable insights.

How might advances in antibody engineering improve the specificity and utility of Phospho-RB1 (S608) detection tools?

Future advances may include:

• Recombinant antibody engineering:

  • Development of single-chain variable fragments (scFvs) for improved tissue penetration

  • Bispecific antibodies targeting both phospho-S608 and total RB1

  • Engineered antibodies with reduced background and increased specificity

• Next-generation detection systems:

  • Proximity ligation assays for studying S608 phosphorylation in situ

  • FRET-based biosensors for live-cell imaging of phosphorylation dynamics

  • Nanobody development for super-resolution microscopy applications

• Intracellular antibodies ("intrabodies"):

  • Development of cell-permeable antibodies for live-cell applications

  • Real-time monitoring of S608 phosphorylation dynamics

  • Targeted modulation of phosphorylated RB1 function

• Multiparametric analysis tools:

  • Mass cytometry-compatible antibodies for high-dimensional analysis

  • Oligonucleotide-conjugated antibodies for spatial transcriptomics integration

  • Multiplexed imaging antibodies for contextual analysis of RB1 phosphorylation

• Computational approaches:

  • Machine learning algorithms to improve phosphorylation pattern recognition

  • Integrative analysis of multiple phosphorylation sites

  • Predictive modeling of phosphorylation dynamics