Phospho-RPS6 (Ser235/236) Antibody

Basic Research Questions

• What is the significance of RPS6 phosphorylation at Ser235/236 versus other phosphorylation sites?

RPS6 (ribosomal protein S6) phosphorylation occurs in an ordered manner, beginning with Ser236 followed sequentially by phosphorylation of Ser235, Ser240, Ser244, and Ser247 . Phosphorylation at Ser235/236 specifically enhances the affinity of RPS6 for the m7GpppG cap complex, promoting translational initiation . While phosphorylation at all sites is often associated with mTORC1 activation, Ser235/236 can also be phosphorylated through the MAPK/RSK pathway, making these sites particularly informative for dissecting signaling crosstalks . Methodologically, this means researchers should consider both mTOR and MAPK inhibitors when studying the specific contributions of each pathway to RPS6 phosphorylation in their experimental system.

• How should I validate Phospho-RPS6 (Ser235/236) antibody specificity for my experiments?

Proper validation should include:

• Lambda protein phosphatase (LPP) treatment of samples to confirm phospho-specificity

• Inclusion of both phosphorylated (stimulated) and non-phosphorylated (starved/inhibitor-treated) control samples

• Primary antibody omission controls to assess non-specific secondary antibody binding

• When possible, use of phospho-deficient RPS6 knockin models (rpS6^P−/−) as negative controls

For immunofluorescence applications, researchers should additionally perform dual staining with total RPS6 antibodies to normalize signals, as demonstrated in subcellular localization studies .

• What experimental controls should I include when using Phospho-RPS6 (Ser235/236) antibodies?

Essential controls include:

For flow cytometry applications, include isotype controls matched to the primary antibody host species and isotype .

Advanced Research Questions

• How can I distinguish between mTORC1 and MAPK/RSK-mediated phosphorylation of RPS6 at Ser235/236?

This distinction requires careful experimental design:

• Perform time-course analyses with pathway-specific inhibitors:

  • Rapamycin or Torin (mTOR inhibitors)

  • U0126 or PD98059 (MEK/ERK pathway inhibitors)

  • BI-D1870 (RSK inhibitor)

• Monitor phosphorylation of:

  • RPS6 at Ser235/236 (can be phosphorylated by both pathways)

  • RPS6 at Ser240/244 (predominantly mTORC1-dependent)

  • ERK1/2 and RSK (MAPK pathway markers)

  • 4E-BP1 (mTORC1-specific substrate)

• Implement genetic approaches using:

  • S6K1/2 knockdown/knockout

  • RSK knockdown/knockout

  • Phospho-deficient RPS6 mutants

TCR stimulation studies have shown that mTOR and ERK/RSK MAPK pathways are partially redundant for RPS6 phosphorylation, with distinct influences on individual serine residues . This requires examination of multiple phospho-sites simultaneously to fully characterize pathway contributions.

• What are the tissue-specific patterns of RPS6 phosphorylation, and how should I optimize immunohistochemistry protocols?

RPS6 phosphorylation patterns vary significantly by tissue:

• In skeletal muscle, phospho-RPS6 shows enhanced peripheral localization following feeding and exercise stimuli, with peripheral-to-central ratios increasing from 1.17 to 1.44 arbitrary units after combined feeding and exercise

• In neuronal tissues, phospho-RPS6 demonstrates differential regulation in specific structures like the nucleus accumbens versus the dorsal striatum

For optimal immunohistochemistry:

• Fixation should be rapid (typically 4% paraformaldehyde) to preserve phospho-epitopes

• Include antigen retrieval steps (often citrate buffer pH 6.0)

• Block with appropriate serum (5-10%) plus BSA (2-5%)

• For quantitative analyses, implement computational methods to assess:

  • Region-specific intensity (central vs. peripheral)

  • Cell-type specific expression when combined with markers

  • Ratio of phospho-to-total RPS6 signal

Antibody concentration should be optimized through serial dilutions (typically 1:300-1:1200 for paraffin sections) .

• How does RPS6 phosphorylation status affect the translation of specific mRNA subsets?

Contrary to earlier assumptions that RPS6 phosphorylation broadly enhances protein synthesis, research using phospho-deficient rpS6^P−/− mice reveals that:

To investigate this phenomenon:

• Perform polysome profiling followed by RNA-seq of heavy polysomal fractions

• Compare translational efficiency between wild-type and phospho-deficient models

• Use Xtail analysis to identify mRNAs with significantly altered translational efficiency

• Validate findings with ribosome profiling or targeted RT-qPCR from polysomal fractions

• What synaptic plasticity changes are associated with altered RPS6 phosphorylation, and how can I measure them?

RPS6 phosphorylation significantly impacts synaptic plasticity in a region-specific manner:

• In phospho-deficient rpS6^P−/− mice:

  • Long-term potentiation (LTP) is specifically impaired in nucleus accumbens D1- and D2-MSNs

  • No changes in LTP are observed in dorsal striatum neurons

  • This occurs despite normal baseline electrophysiological parameters

To assess these changes:

These electrophysiological alterations correlate with behavioral changes such as enhanced novelty-induced locomotion in rpS6^P−/− mice , suggesting functional relevance of these synaptic adaptations.

• How can I optimize flow cytometry protocols for detection of Phospho-RPS6 (Ser235/236)?

Flow cytometry detection of phospho-RPS6 requires special consideration:

• Fixation and permeabilization:

  • Use paraformaldehyde (2-4%) fixation followed by methanol or specialized permeabilization buffers

  • Methanol permeabilization (100%, -20°C, 30 minutes) often yields better results for phospho-epitopes than detergent-based methods

• Antibody selection:

  • Choose directly conjugated antibodies where possible (PE, APC, BV421)

  • For human samples, BioLegend clone A17020B at 1:50-1:200 dilution shows good results

  • For multiplex panels, select fluorophores with minimal spectral overlap

• Sample preparation:

  • Include both stimulated (e.g., PMA/ionomycin) and unstimulated controls

  • For ex vivo samples, immediate fixation is critical to preserve phosphorylation status

  • When analyzing blood lymphocytes, red cell lysis should precede fixation

• Data analysis:

  • Report data as fold-change in median fluorescence intensity

  • Use phospho-flow index (stimulated/unstimulated) for cross-experimental comparisons

  • Consider dimensionality reduction techniques (tSNE, UMAP) for identifying cell populations with differential responses

• What are the mechanisms regulating RPS6 dephosphorylation and how can these be studied?

Despite extensive research on kinases phosphorylating RPS6, the phosphatases involved in dephosphorylation are less characterized:

To study dephosphorylation mechanisms:

• Use phosphatase inhibitors (okadaic acid, calyculin A) at concentrations that differentially inhibit PP1 vs. PP2A

• Perform phosphatase knockdown/knockout studies targeting specific catalytic or regulatory subunits

• Implement phosphatase substrate-trapping mutants to identify direct interactions

• Examine the kinetics of dephosphorylation using pulse-chase approaches with kinase inhibitors

• Consider site-specific dephosphorylation patterns, as mutation of Ser247 inhibits phosphorylation of Ser240/244 but not Ser235/236

Research suggests RPS6 phosphorylation may proceed bidirectionally, with phospho-Ser240/244 promoting Ser247 phosphorylation and vice versa , complicating the analysis of dephosphorylation events.

• What is the relationship between RPS6 phosphorylation and specific neurological or metabolic disorders?

RPS6 phosphorylation has been implicated in several pathological conditions:

• Neurological disorders:

  • In the nucleus accumbens, RPS6 phosphorylation regulates the translation of mitochondria-related mRNAs and affects synaptic plasticity

  • This has implications for motivation, reward, and addiction-related behaviors

  • Altered RPS6 phosphorylation is observed in models of neurodegenerative diseases

• Metabolic disorders:

  • RPS6 is hyperphosphorylated in certain cancers

  • Phosphorylation is a critical determinant of pancreatic β-cell size

  • It influences systemic glucose homeostasis in diabetic mouse models

To investigate these relationships experimentally:

• Use tissue-specific conditional phospho-deficient models

• Perform behavioral and metabolic phenotyping in conjunction with molecular analyses

• Develop phosphorylation site-specific knockin models to dissect the contribution of individual phosphorylation events

• Implement pathway-specific manipulations (e.g., using chemogenetic or optogenetic approaches) to examine acute versus chronic effects of altered RPS6 phosphorylation