Phospho-RPS6 (S235/S236) Antibody

What is ribosomal protein S6 (RPS6) and why is phosphorylation at S235/S236 significant?

RPS6 is a crucial component of the 40S small ribosomal subunit (molecular weight ~32 kDa) with essential roles in controlling cell growth and proliferation through selective translation of particular mRNA classes. The phosphorylation of RPS6 at serines 235 and 236 occurs in response to diverse cellular stimuli including mitogenic stimulation, insulin, and increased nutrient availability. This modification facilitates the recruitment of the 7-methylguanasine cap complex, promoting the assembly of the translational pre-initiation complex and increasing cellular protein synthesis capacity . RPS6 phosphorylation represents a key convergence point for multiple signaling pathways, particularly the mTOR pathway, making it a valuable biomarker for cellular metabolic status and proliferative activity .

RPS6 phosphorylation is highly dynamic and responds rapidly to cellular stimuli. In studies examining dark-to-light transitions in plants, RPS6 phosphorylation increased significantly within 3 hours after light exposure, with polysomal RPS6 showing more dynamic phosphorylation patterns than non-polysomal RPS6 . In mammalian cells, phosphorylation levels can change within minutes following stimulation with growth factors. For example, treatment of HEK293T cells with EGF (100 ng/mL) results in significant phosphorylation of RPS6 within 30 minutes . The phosphorylation status can also show distinctive patterns throughout day-night cycles, with phosphorylation generally peaking during active periods (daytime in plants) and decreasing during rest periods, suggesting integration with metabolic rhythms .

What are the key considerations for selecting an appropriate phospho-RPS6 (S235/S236) antibody?

When selecting a phospho-RPS6 (S235/S236) antibody, researchers should consider:

• Species reactivity: Ensure the antibody recognizes your species of interest. Many commercially available antibodies recognize human, mouse, and rat RPS6 due to high sequence conservation, but verification is essential .

• Specificity: Confirm the antibody specifically detects phosphorylation at S235/S236 and not other phosphorylation sites (S240, S244, S247). Cross-reactivity with these sites can confound results .

• Validated applications: Select antibodies validated for your specific application (Western blot, immunohistochemistry, immunofluorescence, etc.) .

• Clone type: Consider whether a monoclonal or polyclonal antibody better suits your needs. Monoclonal antibodies offer higher specificity but potentially lower sensitivity than polyclonal antibodies .

• Positive controls: Use appropriate positive controls such as EGF-treated cells or tissues known to exhibit high phospho-RPS6 levels .

What are the optimal protocols for detecting phospho-RPS6 (S235/S236) in different applications?

Different applications require specific protocols for optimal results:

Western Blotting:

• Recommended dilution: 1:1000-1:2500

• Sample preparation: Rapidly freeze samples to preserve phosphorylation status

• Buffer system: Use phosphatase inhibitors in lysis buffers to prevent dephosphorylation

• Controls: Include both phosphatase-treated samples and stimulated samples (e.g., EGF-treated cells)

Immunohistochemistry (Paraffin):

• Recommended dilution: 1:300-1:1200

• Antigen retrieval: Critical for detecting phospho-epitopes in fixed tissues

• Fixation: 10% neutral buffered formalin is typically suitable

• Blocking: Use phosphate-free blocking reagents

Immunofluorescence:

• Recommended dilution: 1:800-1:3200

• Fixation: 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100

• Counterstain: DAPI for nuclear visualization

• Controls: Include rapamycin-treated samples as negative controls

How can I distinguish between specific phospho-RPS6 signal and background in my experiments?

To ensure specificity and minimize background:

• Use appropriate negative controls:

  • Samples treated with phosphatase inhibitors versus phosphatase

  • Rapamycin-treated samples (inhibits mTOR signaling)

  • Serum-starved versus serum-stimulated cells

• Validation strategies:

  • Peptide competition assays with phosphorylated versus non-phosphorylated peptides

  • Genetic knockdown/knockout of RPS6 or upstream kinases

  • Site-directed mutagenesis (S235A/S236A) to eliminate phosphorylation sites

• Signal verification:

  • Compare signal patterns with total RPS6 antibody staining

  • Verify molecular weight (~32 kDa) in Western blots

  • Confirm expected subcellular localization (primarily cytoplasmic)

How can phospho-RPS6 (S235/S236) be used as a biomarker in cancer research?

Phospho-RPS6 serves as a valuable biomarker in cancer research for several reasons:

• Pathway activation indicator: Phospho-RPS6 levels reflect activation of mTOR and MAPK pathways, which are frequently dysregulated in cancer .

• Therapeutic response monitoring: Changes in RPS6 phosphorylation can indicate response to targeted therapies. For example, in trastuzumab-resistant breast cancer cells, p-RPS6 levels remain unchanged following treatment, unlike in sensitive cells where phosphorylation decreases .

• Correlation with proliferation: Phospho-RPS6 levels correlate with Ki67 status, providing insights into tumor proliferation rates .

• Resistance mechanism identification: In HER2-positive breast cancer, persistent p-RPS6 expression following treatment may indicate bypass mechanisms of resistance, as demonstrated in BT474R and SKBR3R resistant cell lines .

• Predictive biomarker: The inverse correlation between p-RPS6 levels and growth inhibition in drug-resistant cells makes it a potential predictor of response to HER2-targeting agents .

What role does RPS6 phosphorylation play in neuronal function and neuroscience research?

RPS6 phosphorylation plays significant roles in neuronal function:

• Novelty response: Phospho-RPS6 is involved in novelty-induced translation of specific mRNAs in the nucleus accumbens (Acb), but not in the dorsal striatum, indicating region-specific functions in the brain .

• Protein synthesis regulation: In neurons, RPS6 phosphorylation modulates local protein synthesis, which is crucial for synaptic plasticity, memory formation, and learning .

• Stimulus-dependent translation: RPS6 phosphorylation controls the translation of specific subsets of mRNAs rather than global translation in brain regions like the Acb, as demonstrated through polysome profiling and RNAseq analysis .

• Regional specificity: Research in rpS6 P−/− mice revealed 998 differentially expressed mRNAs in the Acb polysomal fraction without changes in total mRNA abundance, indicating translational rather than transcriptional regulation .

• Signaling integration: In neurons, RPS6 phosphorylation integrates various signaling inputs, including dopaminergic, glutamatergic, and neuromodulatory signals, making it a hub for neuronal activity-dependent translation .

How does phospho-RPS6 integrate with circadian rhythms and metabolic regulation?

Recent research has revealed interesting connections between RPS6 phosphorylation, circadian rhythms, and metabolism:

• Diel phosphorylation cycles: RPS6 phosphorylation shows reproducible 24-hour cycles with peaks during active periods and troughs around dawn, suggesting integration with circadian mechanisms .

• Light-dark integration: In plants, RPS6 phosphorylation increases rapidly upon dark-to-light transitions, indicating responsiveness to environmental cues that regulate metabolism .

• Polysome association changes: The distribution of phosphorylated RPS6 between non-polysomal, small polysomal, and large polysomal fractions changes throughout the day, with polysomal RPS6 showing more dynamic phosphorylation patterns .

• Metabolic regulation: In mammalian systems, RPS6 phosphorylation status affects glucose homeostasis and pancreatic β-cell size, as demonstrated in diabetic mouse models .

• Nutrient sensing: RPS6 phosphorylation serves as a convergence point for nutrient availability signals and growth factor inputs, allowing cells to coordinate protein synthesis with metabolic status .

How can I differentiate between the contributions of different upstream kinases to RPS6 phosphorylation?

To distinguish between kinase contributions:

• Pharmacological inhibitors:

  • mTOR pathway: Rapamycin (50 nM) or Torin1

  • MEK/ERK pathway: U0126 or PD98059

  • PI3K/Akt pathway: Wortmannin or LY294002

  • PKA: H-89 or PKI

• Genetic approaches:

  • RNAi knockdown of specific kinases

  • CRISPR/Cas9 knockout of kinase genes

  • Dominant-negative kinase mutants

  • Constitutively active kinase constructs

• Stimulus-specific activation:

  • Growth factors for mTOR activation (EGF, insulin)

  • cAMP-elevating agents for PKA (forskolin)

  • Stress stimuli for MAPK/RSK

• Combined inhibitor approaches: Using inhibitor combinations can reveal pathway cross-talk and compensation mechanisms. For example, T cell studies have shown that both mTOR and MAPK pathways contribute to RPS6 phosphorylation .

• Time-course analysis: Different kinases may contribute differently depending on the timing after stimulation, making temporal analysis valuable .

What approaches can I use to study the functional consequences of RPS6 phosphorylation on protein synthesis?

Several sophisticated approaches can link RPS6 phosphorylation to protein synthesis regulation:

• Polysome profiling with phospho-specific detection:

  • Separate ribosomes based on translational activity

  • Analyze phospho-RPS6 distribution across fractions

  • Compare polysome profiles under different conditions

• Ribosome footprinting with phospho-RPS6 immunoprecipitation:

  • Isolate ribosome-protected fragments

  • Compare mRNAs associated with phosphorylated versus non-phosphorylated RPS6

• Knock-in models with phospho-mutants:

  • Generate S235A/S236A phospho-deficient mutants

  • Create phosphomimetic mutations (S235D/S236D)

  • Analyze translation patterns in these models

• Translational efficiency measurements:

  • SUnSET (surface sensing of translation) to measure protein synthesis rates

  • [35S] methionine/cysteine labeling followed by immunoprecipitation

  • Nascent proteomics approaches

• mRNA-specific translation analysis:

  • RNA immunoprecipitation of specific mRNAs with phospho-RPS6

  • TRAP (translating ribosome affinity purification) combined with phospho-specific analysis

How can I investigate the spatial and temporal dynamics of RPS6 phosphorylation in living cells?

To study real-time dynamics of RPS6 phosphorylation:

• Phospho-specific FRET biosensors:

  • Design FRET sensors that respond to RPS6 phosphorylation

  • Monitor phosphorylation in real-time in living cells

  • Analyze subcellular compartment-specific phosphorylation events

• Live-cell immunofluorescence with cell-permeable antibodies:

  • Utilize cell-permeable versions of phospho-RPS6 antibodies

  • Track phosphorylation changes in response to stimuli

• Optogenetic control of upstream kinases:

  • Use light-activated kinases to induce phosphorylation

  • Determine spatial and temporal resolution of phosphorylation events

• Fluorescence correlation spectroscopy (FCS):

  • Analyze diffusion coefficients of phosphorylated versus non-phosphorylated RPS6

  • Determine association with other proteins or complexes

• Proximity ligation assays in fixed time-course samples:

  • Visualize interactions between phospho-RPS6 and binding partners

  • Map subcellular localization of interactions

What are common issues when detecting phospho-RPS6 (S235/S236) and how can they be resolved?

How should I interpret discrepancies between different phospho-RPS6 antibodies or detection methods?

When facing discrepancies:

• Epitope recognition differences:

  • Some antibodies may recognize multiple phosphorylation sites

  • Confirm epitope specificity using phosphopeptide competition

  • Use site-specific antibodies in parallel to verify results

• Method sensitivity variations:

  • Western blotting may detect lower levels than immunohistochemistry

  • Flow cytometry provides single-cell resolution but different sensitivity

  • ELISA may offer quantitative advantages but lacks spatial information

• Resolution of conflicting data:

  • Validate with multiple antibodies from different sources

  • Employ orthogonal detection methods

  • Use genetic models (phospho-mutants) as definitive controls

  • Consider timing differences in sample collection

• Technical considerations:

  • Different buffers may affect epitope accessibility

  • Fixation methods vary in their preservation of phospho-epitopes

  • Antibody dilutions should be optimized for each application

What are the best approaches for quantifying phospho-RPS6 (S235/S236) levels in complex samples?

For accurate quantification:

• Normalization strategies:

  • Normalize to total RPS6 levels rather than housekeeping proteins

  • Use phosphorylation-independent epitopes on RPS6 as internal controls

  • Include loading controls appropriate for the subcellular fraction analyzed

• Quantitative methods:

  • Western blot with standard curves using recombinant phosphorylated proteins

  • ELISA assays specifically designed for phospho-RPS6 quantification

  • Mass spectrometry-based approaches for absolute quantification

• Single-cell analysis:

  • Flow cytometry for heterogeneous populations

  • Imaging cytometry for adherent cells

  • Single-cell Western blotting for rare cell types

• Multiplexed detection:

  • Simultaneous measurement of multiple phosphorylation sites

  • Co-detection of upstream and downstream pathway components

  • Correlation with functional readouts (e.g., proliferation, protein synthesis)

• Specialized techniques for tissues:

  • Laser capture microdissection to isolate specific cell types

  • Spatial transcriptomics combined with phospho-protein mapping

  • Tissue clearing and 3D imaging of phospho-epitopes

How can phospho-RPS6 antibodies be used to investigate translational control in disease models?

Phospho-RPS6 antibodies offer valuable insights into translational dysregulation in disease:

• Cancer models:

  • Monitoring treatment response in drug-resistant tumors

  • Identifying tumors likely to respond to mTOR inhibitors

  • Analyzing pathway activation in patient-derived xenografts

• Neurodegenerative diseases:

  • Investigating protein synthesis alterations in Alzheimer's or Parkinson's disease models

  • Examining stress responses in affected neurons

  • Monitoring effects of therapeutic interventions on translation

• Metabolic disorders:

  • Studying insulin resistance effects on protein synthesis

  • Analyzing nutrient sensing defects in obesity models

  • Investigating pancreatic β-cell adaptation to metabolic stress

• Immunological research:

  • Tracking T cell activation through phospho-RPS6 dynamics

  • Studying translational responses during immune challenge

  • Monitoring cellular stress responses during inflammation

What novel methodological approaches combine phospho-RPS6 detection with other advanced techniques?

Innovative methodological combinations include:

• Spatial transcriptomics with phospho-proteomics:

  • Map spatial distribution of phospho-RPS6 alongside mRNA profiles

  • Correlate localized translation with phosphorylation status

  • Identify microenvironmental factors influencing RPS6 phosphorylation

• Single-cell phospho-proteomics:

  • Analyze cell-to-cell variation in phospho-RPS6 levels

  • Correlate with other signaling pathways at single-cell resolution

  • Identify rare cell populations with unique translational profiles

• CRISPR screens with phospho-RPS6 readouts:

  • Identify novel regulators of RPS6 phosphorylation

  • Screen for synthetic lethal interactions with phospho-RPS6 dependencies

  • Discover pathway components using phospho-RPS6 as a phenotypic marker

• In vivo imaging of phospho-RPS6:

  • Develop phospho-specific reporters for in vivo imaging

  • Track temporal dynamics during development or disease progression

  • Monitor therapeutic responses in real-time

• Computational modeling of phosphorylation networks:

  • Integrate phospho-RPS6 data into signaling network models

  • Predict intervention points for modulating translation

  • Model kinase-phosphatase balance in different cellular contexts