Phospho-RB1 (S612) Antibody

What is the biological significance of RB1 S612 phosphorylation in cell cycle regulation?

The phosphorylation of RB1 at S612 is one of the key modifications that regulates its function as a cell cycle inhibitor. Located in the pocket domain, S612 phosphorylation has been mechanistically proven to strongly affect RB-E2F interactions and is therefore strongly implicated in abrogating RB's tumor suppressive capacity .

RB1 exists in different phosphorylation states throughout the cell cycle:

• Unphosphorylated form: Predominantly in G0 phase (quiescent cells)

• Mono-phosphorylated forms: Present in early G1 phase

• Hyper-phosphorylated forms: Appear in late G1 and S phases

S612 can exist as one of the 14 independent mono-phosphorylated RB isoforms in early G1 phase, each potentially having unique functions . This mono-phosphorylation is primarily mediated by cyclin D:Cdk4/6 complexes .

How does mono-phosphorylation at S612 differ functionally from other RB1 phosphorylation sites?

Each mono-phosphorylated RB isoform, including S612, appears to have distinct functional properties. Research has shown that individual mono-phosphorylated RB isoforms form different protein complexes with varying compositions, suggesting site-specific functional specialization .

The functional specificity of different mono-phosphorylated RB isoforms extends beyond cell cycle regulation. For example, while S811 phosphorylation promotes association with the NuRD chromatin-remodeling complex and alters gene repression patterns, other sites like S612 may regulate different protein-protein interactions and cellular processes .

Experimental data shows that all 14 mono-phosphorylated RB isoforms are capable of arresting cells in G1-phase, confirming they remain active as cell cycle regulators, but with varying efficiencies. For instance, T356 and S788 mono-phosphorylation resulted in the greatest G1 increase .

What techniques are available for detecting RB1 S612 phosphorylation?

Several techniques can be employed to detect RB1 S612 phosphorylation:

For optimal results, researchers should use phospho-specific antibodies that recognize RB1 only when phosphorylated at S612. These antibodies are typically generated using synthetic phosphopeptides corresponding to residues surrounding S612 of human RB1 .

What are the optimal conditions for using Phospho-RB1 (S612) antibody in Western blot analysis?

For optimal Western blot results with Phospho-RB1 (S612) antibody:

• Sample preparation:

  • Use fresh cell lysates or properly stored frozen samples

  • Include phosphatase inhibitors in lysis buffer to prevent dephosphorylation

  • Normalize protein loading (30-50 μg total protein per lane recommended)

• Antibody conditions:

  • Typical dilution: 1:1000 for Western blot applications

  • Prepare antibody solution in TBST with 5% BSA and 0.02% sodium azide

  • Incubate membrane with primary antibody overnight at 4°C

• Controls:

  • Include both phosphorylated (e.g., S-phase arrested cells) and unphosphorylated (G0 arrested cells) controls

  • Blot for total RB1 on the same membrane after stripping or on a parallel membrane

  • Report results as the ratio of phosphorylated RB1 to total RB1 protein

• Troubleshooting:

  • High background may indicate contaminated antibody solution; replace with fresh preparation

  • Check for cross-reactivity with other phosphorylation sites (antibodies should be validated for specificity)

How can I validate the specificity of Phospho-RB1 (S612) antibody in my experiments?

Validating specificity is crucial for phospho-specific antibodies. Several approaches include:

• Phosphatase treatment: Treat one sample with lambda phosphatase before immunoblotting. The signal should disappear in the treated sample if the antibody is specific for the phosphorylated form.

• Phospho-blocking peptide: Pre-incubate the antibody with the phosphopeptide used as immunogen. This should block specific binding and eliminate the signal.

• Mutant controls: Use cell lines expressing RB1 with a serine-to-alanine mutation at position 612 (S612A) as a negative control.

• Cross-reactivity testing: Test against other phosphorylated residues on RB1. For example, the Phospho-Rb (Ser807/811) antibody specifically doesn't cross-react with RB phosphorylated at Ser608 , and similar specificity should be confirmed for S612 antibodies.

• Cell cycle synchronization: Compare G0-arrested cells (unphosphorylated RB) versus S-phase cells (hyperphosphorylated RB) to confirm the antibody detects the expected differences in phosphorylation patterns.

What are the best sample preparation methods for detecting RB1 S612 phosphorylation?

For optimal detection of RB1 S612 phosphorylation:

• Cell synchronization:

  • For unphosphorylated RB1: Serum deprivation (G0 arrest)

  • For mono-phosphorylated RB1: Contact inhibition (early G1 arrest)

  • For hyper-phosphorylated RB1: S-phase arrest using thymidine block or aphidicolin

• Lysis conditions:

  • Use fresh samples whenever possible

  • Include both phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) and protease inhibitors

  • Maintain cold temperature throughout sample preparation

  • Use buffers with neutral to slightly alkaline pH (7.4-8.0)

• Storage:

  • Aliquot samples to avoid repeated freeze-thaw cycles

  • Store at -80°C for long-term preservation

  • Include glycerol (10-20%) in storage buffer

• Normalization:

  • Always quantify total protein concentration

  • Include loading controls (e.g., β-actin, GAPDH)

  • Blot for total RB1 protein to calculate phosphorylation ratio

How can I distinguish between mono-phosphorylated and hyper-phosphorylated forms of RB1?

Distinguishing between mono-phosphorylated and hyper-phosphorylated RB1 requires specific techniques:

• Two-dimensional isoelectric focusing (2D IEF): This technique separates proteins based on charge and can resolve different phosphorylation states. Mono-phosphorylated RB shows a distinct pattern compared to hyper-phosphorylated RB .

• Sequential immunoprecipitation: First, immunoprecipitate with a phospho-specific antibody (e.g., S612), then immunoblot with multiple phospho-specific antibodies. For mono-phosphorylated RB, only the immunoprecipitating antibody will detect the protein, whereas hyper-phosphorylated RB will be detected by multiple phospho-specific antibodies .

• Mobility shift: On standard SDS-PAGE, hyper-phosphorylated RB migrates more slowly than mono-phosphorylated or unphosphorylated forms.

• Timing during cell cycle:

  • G0: Unphosphorylated RB

  • Early G1: Mono-phosphorylated RB (including S612)

  • Late G1/S: Hyper-phosphorylated RB

Research has shown that the phospho-specific immunoprecipitation of mono-phosphorylated RB from early G1 phase cells is only recognized by immunoblot with the same phospho-specific antibody and not by other phospho-specific antibodies. In contrast, immunoprecipitated hyper-phosphorylated RB from S phase arrested cells is recognized by multiple phospho-specific antibodies .

What are the implications of detecting RB1 S612 phosphorylation in cancer samples?

The detection of RB1 S612 phosphorylation in cancer samples has several important implications:

• Diagnostic value: Phosphorylation at S612 (located in the pocket domain) strongly affects RB-E2F interactions and is therefore implicated in abrogating RB's tumor suppressive capacity. Its detection can provide valuable diagnostic information .

• Prognostic significance: Hyperphosphorylation of RB has been associated with pathological states such as cancer. Assessing specific phosphorylation sites like S612 may provide prognostic value .

• Therapeutic targeting: Understanding the phosphorylation pattern of RB1 in specific cancers can inform therapeutic decisions, particularly for therapies targeting cell cycle regulators like CDK4/6 inhibitors .

• Resistance mechanisms: Changes in RB1 phosphorylation patterns may indicate development of resistance to CDK inhibitors or other targeted therapies.

• Molecular etiology: For cancer biologists, examining S612 phosphorylation status is an important tool for understanding the molecular etiology of cancer and how RB1 function is compromised .

Research has shown that phosphorylations occurring in the pocket domain (like S612) and C-terminal domain are extremely informative regarding the oncogenic proclivity of a cell, and when conducted in human tumor samples, can provide valuable diagnostic, prognostic and therapy responsiveness information .

How do different mono-phosphorylated RB isoforms interact with other cellular proteins?

Research has revealed significant differences in protein interactions among the various mono-phosphorylated RB isoforms:

• E2F transcription factors: Different mono-phosphorylated RB isoforms show variable binding to E2F family members (E2F-1, E2F-2, E2F-3, E2F-4). This suggests a mechanism by which RB can regulate specific E2F-responsive genes differently based on its phosphorylation state .

• Viral oncoproteins: For example, the E1a oncoprotein from adenovirus was found to bind equally well to unphosphorylated RB and all 14 mono-phosphorylated RB isoforms, suggesting that viral proteins have evolved to overcome all forms of RB regulation .

• Chromatin modifiers: Some specific mono-phosphorylated forms have been shown to preferentially interact with chromatin-remodeling complexes. For instance, RB phosphorylation at S811 promotes association with the NuRD chromatin-remodeling complex and alters the spectrum of genes repressed by RB .

• Mitochondrial proteins: Proteomic studies have indicated that mitochondrial changes are a major consequence of RB inactivation, suggesting that some mono-phosphorylated forms may regulate mitochondrial function .

Research has demonstrated that the specific mono-phosphorylation event provides functional specificity that extends beyond the regulation of the cell cycle, with significant differences in the composition of protein complexes formed by individual mono-phosphorylated RB isoforms .

How should I design experiments to investigate the functional consequences of S612 phosphorylation?

To investigate the functional consequences of S612 phosphorylation:

• Phospho-mimetic and phospho-deficient mutants:

  • Generate S612D or S612E (phospho-mimetic) and S612A (phospho-deficient) RB1 mutants

  • Express these in RB1-null cell lines to isolate the effect of this specific phosphorylation

• Inducible systems:

  • Develop isogenic cell lines with inducible expression of wild-type or mutant RB1

  • This approach has been used successfully to study mono-phosphorylated RB isoforms

• Proteomic analysis:

  • Perform immunoprecipitation followed by mass spectrometry to identify proteins that interact specifically with S612-phosphorylated RB1

  • Compare interaction partners between different phosphorylation states

• Transcriptomic analysis:

  • Compare gene expression profiles in cells expressing wild-type RB1 versus S612A or S612D mutants

  • Focus on E2F-responsive genes and pathways

• Cell cycle analysis:

  • Assess impact on G1/S transition using flow cytometry

  • Compare with other mono-phosphorylated RB isoforms, as research has shown that all 14 mono-phosphorylated RBs arrested cells in G1-phase but with varying efficiency

• Kinase manipulation:

  • Use specific CDK4/6 inhibitors (e.g., PD0332991) or expression of p16 to prevent S612 phosphorylation

  • Assess downstream effects on cell cycle progression and gene expression

Studies have successfully used these approaches to demonstrate that different mono-phosphorylated RB isoforms have distinct activities and protein interaction partners .

What controls are essential when analyzing RB1 phosphorylation at S612?

Essential controls for RB1 S612 phosphorylation analysis include:

• Phosphorylation state controls:

  • Unphosphorylated RB: Serum-starved G0 cells or cells treated with CDK4/6 inhibitors

  • Hyper-phosphorylated RB: S-phase synchronized cells

  • Cells expressing phospho-deficient RB (S612A mutant)

• Antibody controls:

  • Phosphatase-treated samples to confirm phospho-specificity

  • Blocking peptide competition assays

  • Secondary antibody-only controls to assess non-specific binding

• Total RB1 quantification:

  • Always blot for total RB protein to calculate the phosphorylation ratio

  • This is critical as the extent of RB phosphorylation should be assessed and reported as the ratio of phosphorylated RB to total RB protein

• Cell cycle controls:

  • Synchronize cells at different cell cycle phases

  • Confirm cell cycle phase by flow cytometry or by blotting for cell cycle markers

• Kinase manipulation:

  • CDK4/6 inhibitor (e.g., PD0332991) treatment as a negative control

  • p16 expression to specifically inhibit cyclin D:Cdk4/6 complexes

Research has shown that treatment with CDK4/6 inhibitors or expression of p16 results in the presence of unphosphorylated RB, confirming cyclin D:Cdk4/6 as the RB mono-phosphorylating kinase in vivo .

How can phospho-RB1 (S612) antibody be used in multi-parameter analyses?

Phospho-RB1 (S612) antibody can be incorporated into various multi-parameter analyses:

• Multiplexed immunofluorescence:

  • Combine with antibodies against other phosphorylation sites on RB1

  • Co-stain with cell cycle markers (e.g., Ki67, PCNA)

  • Include markers for specific cellular compartments to assess localization

• Flow cytometry:

  • Conjugate phospho-RB1 (S612) antibody with fluorescent dyes like Alexa Fluor 555

  • Combine with DNA content staining to correlate phosphorylation with cell cycle phase

  • Use with other intracellular markers for multi-parameter analysis

• Proximity ligation assay (PLA):

  • Detect interactions between S612-phosphorylated RB1 and suspected binding partners

  • Provides spatial information about these interactions in situ

• ChIP-seq analysis:

  • Use phospho-RB1 (S612) antibody for chromatin immunoprecipitation

  • Identify genomic regions bound by this specific phosphorylated form

  • Compare with binding profiles of other phosphorylated forms

• Proteomics approaches:

  • Immunoprecipitate with phospho-RB1 (S612) antibody followed by mass spectrometry

  • Identify differential protein complexes formed specifically with S612-phosphorylated RB1

This multi-parameter approach has been used successfully to demonstrate that mono-phosphorylated RB isoforms have distinct activities and protein interaction partners. For example, RB phosphorylation at S811 promotes association with the NuRD chromatin-remodeling complex and alters gene repression patterns .

By integrating these approaches, researchers can gain comprehensive insights into the specific functions of S612 phosphorylation in various cellular contexts.

What are common issues when detecting RB1 S612 phosphorylation and how can they be resolved?

Common issues and their solutions include:

• High background in immunoblotting:

  • Problem: Non-specific binding or contaminated antibody solutions

  • Solution: Use fresh antibody solutions, increase blocking time/concentration, optimize antibody dilution, include Tween-20 in wash buffers

• Weak or absent signal:

  • Problem: Low phosphorylation levels or dephosphorylation during sample preparation

  • Solution: Include phosphatase inhibitors, avoid prolonged sample handling at room temperature, optimize cell synchronization to capture peak phosphorylation

• Multiple bands or unexpected band size:

  • Problem: Degradation products or cross-reactivity

  • Solution: Use fresh samples with protease inhibitors, validate antibody specificity, include positive controls

• Inconsistent results between experiments:

  • Problem: Variable phosphorylation levels due to cell cycle asynchrony

  • Solution: Carefully synchronize cells, standardize culture conditions, include cell cycle markers as controls

• Poor reproducibility in phosphorylation quantification:

  • Problem: Technical variation in immunoblotting and detection

  • Solution: Use the ratio of phosphorylated RB to total RB protein rather than absolute phosphorylation levels

Research has shown that use of contaminated antibody solution usually yields high background, meaning that it is time to replace the solution with a fresh one .

How do cell cycle synchronization methods affect the detection of S612 phosphorylation?

Different synchronization methods significantly impact the detection of RB1 S612 phosphorylation:

• Serum deprivation (G0 arrest):

  • Results in unphosphorylated RB

  • Useful as a negative control for phospho-specific antibodies

  • None of the phospho-specific RB antibodies recognize unphosphorylated RB from G0 arrested cells

• Contact inhibition (early G1 arrest):

  • Results in mono-phosphorylated RB isoforms, including S612

  • Ideal for studying individual mono-phosphorylated forms

  • All phospho-specific RB antibodies recognize mono-phosphorylated RB isoforms from early G1 phase arrested cells

• CDK4/6 inhibition:

  • Treatment with PD0332991 (a selective CDK4/6 inhibitor) or expression of p16 prevents RB phosphorylation

  • Results in unphosphorylated RB similar to G0 arrest

  • Confirms that cyclin D:Cdk4/6 is the RB mono-phosphorylating kinase

• S-phase arrest (thymidine block or aphidicolin):

  • Results in hyper-phosphorylated RB

  • All phospho-specific antibodies recognize hyper-phosphorylated RB from S phase arrested cells

  • Useful as a positive control for phospho-specific antibodies

To accurately assess S612 phosphorylation across the cell cycle, researchers should combine these synchronization methods with precise timing of sample collection.