Phospho-RB1 (T826) Antibody

What is the biological significance of RB1 phosphorylation at threonine 826?

Phosphorylation of RB1 at threonine 826 (T826) represents a critical post-translational modification in cell cycle regulation. This specific phosphorylation event is part of the sequential phosphorylation pattern that occurs during the G1/S transition. Research indicates that T826 phosphorylation typically occurs in early G1 phase, alongside other sites including S249, T252, T356, S608, S788, S807, and S811 . The cumulative effect of these phosphorylation events contributes to RB1's dissociation from E2F transcription factors, enabling E2F-responsive gene expression and cell cycle progression . When designing experiments to study cell cycle progression, monitoring T826 phosphorylation provides a specific readout of CDK activity and cell cycle position.

What detection methods are validated for Phospho-RB1 (T826) antibodies?

Phospho-RB1 (T826) antibodies have been validated for multiple detection methods:

For optimal results, researchers should include appropriate controls such as phosphatase-treated samples and total RB1 detection in parallel experiments .

How can researchers properly validate the specificity of Phospho-RB1 (T826) antibodies?

Researchers should implement multiple validation strategies:

• Phosphatase treatment: Samples should be divided and one portion treated with lambda phosphatase to demonstrate loss of signal with the phospho-specific antibody

• Cell cycle synchronization: Compare cells arrested in G0/G1 (minimal phosphorylation) with proliferating cells

• RB1 knockdown/knockout controls: Confirm absence of signal in RB1-depleted cells

• Peptide competition assays: Pre-incubation of antibody with phosphorylated and non-phosphorylated peptides should show specific blocking only with the phosphorylated form

• Comparison with total RB1 detection: Use both phospho-specific and total RB1 antibodies to determine the proportion of phosphorylated protein

Following these methodological approaches ensures experimental rigor and reproducibility when studying this specific post-translational modification.

How does the SETDB1-TRIM28 axis regulate phosphorylated RB1 stability?

Recent research has uncovered a sophisticated regulatory mechanism controlling phosphorylated RB1 stability involving SETDB1 and TRIM28:

• TRIM28-mediated degradation: The RING finger domain protein TRIM28 specifically binds to CDK4/6-phosphorylated RB1 and promotes its ubiquitination, targeting it for proteasomal degradation

• SETDB1 protective function: SETDB1 counteracts this process by:

  • Binding to methylated RB1 through its Tudor domain

  • Protecting phosphorylated RB1 from TRIM28-mediated degradation

  • Functioning independently of its methyltransferase activity

• Clinical implications: SETDB1 is frequently overexpressed due to gene amplification in prostate cancer and positively correlates with phosphorylated RB1 levels in patient specimens

• Therapeutic potential: Combined inhibition of SETDB1 and CDK4/6 has shown synergistic effects in cancer models:

  • SETDB1 inhibition with antisense oligonucleotides reduces tumor growth but accelerates RB1 degradation

  • Co-administration of CDK4/6 inhibitor (palbociclib) blocks this degradation and enhances therapeutic efficacy

This regulatory pathway highlights how post-translational modifications beyond phosphorylation (i.e., methylation) can affect RB1 function and stability in cancer contexts.

What are the technical challenges in studying the temporal sequence of RB1 phosphorylation at multiple sites?

Studying the complex phosphorylation patterns of RB1 presents several methodological challenges:

• Temporal resolution limitations: Standard immunoblotting techniques may not provide sufficient temporal resolution to precisely map phosphorylation kinetics

• Antibody cross-reactivity concerns: When multiple phosphorylation sites are in proximity, antibody cross-reactivity must be rigorously tested

• Mass spectrometry approaches:

  • Phosphoproteomic analysis has confirmed 13 of 16 consensus CDK sites in human RB1

  • Studies in synchronized HeLa cells revealed extensive RB1 phosphorylation in M phase, but only S249 and T252 phosphorylation in G1

• Mutational analysis limitations: Site-directed mutagenesis approaches (e.g., T826A) may disrupt protein folding or other phosphorylation events

• Cell synchronization artifacts: Methods to synchronize cells may introduce artifacts in phosphorylation patterns

Researchers should employ complementary approaches, including phospho-specific antibodies, mass spectrometry, and genetic models with site-specific mutations to comprehensively map the temporal dynamics of RB1 phosphorylation.

How does cytoplasmic versus nuclear localization affect phosphorylated RB1 (T826) detection and function?

Recent studies have revealed distinct functional implications for cytoplasmic versus nuclear phosphorylated RB1:

• Cytoplasmic translocation mechanisms:

  • TNF-α treatment can induce phosphorylation and cytoplasmic accumulation of RB1

  • Phosphorylated cytoplasmic RB1 interacts with different binding partners than nuclear RB1

• Non-nuclear functions:

  • Cytoplasmic RB1 interacts with mDia1 through its LXCXE motif contained in the GTPase binding domain

  • This interaction may disrupt sarcomeric organization in muscle cells

• Detection considerations:

  • Subcellular fractionation is essential when quantifying phosphorylated RB1

  • Immunofluorescence with phospho-specific antibodies can reveal localization patterns

  • Use of NES-tagged RB1 constructs allows experimental manipulation of localization

• Experimental design recommendations:

  • Include cytoplasmic and nuclear markers in immunofluorescence experiments

  • Perform subcellular fractionation prior to immunoblotting

  • Consider cell type-specific differences in RB1 localization patterns

Understanding the distinct roles of nuclear versus cytoplasmic phosphorylated RB1 provides insights into non-canonical functions beyond cell cycle regulation.

What methodological approaches can resolve contradictory findings regarding the role of T826 phosphorylation in cancer progression?

Researchers encountering contradictory findings about T826 phosphorylation should consider these methodological approaches:

• Cell type and context specificity:

  • Systematically compare multiple cell lines and primary tissues

  • Evaluate the impact of genetic background on phosphorylation patterns

  • Develop isogenic cell line models to isolate the effect of specific mutations

• Temporal dynamics assessment:

  • Use real-time imaging with fluorescent reporters of RB1 phosphorylation

  • Implement synchronized cell populations with minimal perturbation

  • Consider single-cell analysis techniques to capture heterogeneity

• Systems biology integration:

  • Correlate T826 phosphorylation with other post-translational modifications

  • Develop mathematical models of phosphorylation dynamics

  • Integrate transcriptomic data to link phosphorylation states with gene expression

• In vivo validation:

  • Generate knock-in mouse models with phosphomimetic or phospho-dead mutations

  • Develop PDX models retaining tumor heterogeneity

  • Employ tissue-specific conditional expression systems

• Therapeutic intervention studies:

  • Test the effect of CDK4/6 inhibitors on T826 phosphorylation in different contexts

  • Evaluate SETDB1-TRIM28 axis modulation as described in recent findings

  • Investigate combination approaches targeting multiple regulatory mechanisms

These approaches enable researchers to dissect the functional significance of T826 phosphorylation while accounting for biological complexity and technical variability.

How can multiplexed detection methods be optimized for simultaneous analysis of multiple RB1 phosphorylation sites?

Developing robust multiplexed detection approaches for RB1 phosphorylation requires:

• Multiplex immunofluorescence optimization:

  • Careful antibody selection to avoid species cross-reactivity

  • Sequential staining protocols to minimize antibody interference

  • Spectral unmixing to resolve overlapping fluorophores

  • Inclusion of phospho-T826 with other key sites (S608, S795, T821)

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated antibodies for higher multiplexing capacity

  • Combined detection of phospho-RB1 sites with cell cycle markers

  • Single-cell resolution of RB1 phosphorylation states

• Microwestern array development:

  • Miniaturized western blotting for multiple phospho-sites

  • Quantitative comparison across experimental conditions

  • Higher throughput screening of inhibitor effects

• Phospho-proteomics integration:

  • Targeted mass spectrometry for quantitative phospho-site analysis

  • SILAC labeling for comparative studies

  • Correlation of T826 phosphorylation with global phosphoproteome changes

• Validation strategies:

  • Sequential probing of the same membrane with different phospho-antibodies

  • Parallel processing of identical samples with different antibodies

  • Use of phosphatase treatments as negative controls