Phospho-RPS6KB1 (T412) Antibody

What is RPS6KB1 and what does the phosphorylation at T412 signify?

RPS6KB1 (Ribosomal Protein S6 Kinase, 70kDa, Polypeptide 1), also known as p70 S6 Kinase or S6K1, is a serine/threonine-protein kinase that functions downstream of mTOR signaling in response to growth factors and nutrients. This kinase plays crucial roles in promoting cell proliferation, growth, and cell cycle progression .

Phosphorylation at threonine 412 (T412) is a critical regulatory modification that signifies activation of the kinase. This site is regulated by mTORC1, and the phosphorylation is maintained by an agonist-dependent autophosphorylation mechanism . When phosphorylated at T412, the kinase becomes activated and can subsequently phosphorylate its downstream targets, including ribosomal protein S6, EIF4B, and EEF2K, which are involved in protein synthesis and translation .

What are the key applications for Phospho-RPS6KB1 (T412) antibodies in research?

Phospho-RPS6KB1 (T412) antibodies are utilized in multiple experimental applications:

• Western Blotting (WB): For detecting endogenous levels of phosphorylated RPS6KB1 in cell or tissue lysates, typically at dilutions ranging from 1:500-1:2000

• Immunohistochemistry (IHC): For visualizing the localization of phosphorylated RPS6KB1 in tissue sections, commonly used at dilutions of 1:50-1:300

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For subcellular localization studies in cultured cells, with recommended dilutions of 1:200-1:1000

• ELISA: For quantitative measurement of phosphorylated RPS6KB1 levels, often at high dilutions around 1:10000

• Immunoprecipitation (IP): For isolating phosphorylated RPS6KB1 complexes from cell lysates

Each application requires specific optimization for the particular experimental conditions and cell/tissue types being studied.

How should researchers store and handle Phospho-RPS6KB1 (T412) antibodies to maintain reactivity?

Proper storage and handling of Phospho-RPS6KB1 (T412) antibodies is essential for maintaining their reactivity and specificity:

• Aliquot the antibody upon receipt to minimize freeze-thaw cycles

• Avoid repeated freeze-thaw cycles as they can degrade the antibody

• Briefly centrifuge the antibody vial before opening to collect the solution at the bottom

• When diluting for experiments, use fresh, cold buffer systems appropriate for the application

Temperature control during shipping and storage is critical, as noted in several product specifications .

What controls should be included when using Phospho-RPS6KB1 (T412) antibodies?

A rigorous experimental design should include the following controls when working with Phospho-RPS6KB1 (T412) antibodies:

Positive Controls:

• Cell lysates from cells treated with activators of the mTOR pathway (insulin, growth factors)

• Recombinant phosphorylated RPS6KB1 protein standards

• Previously validated positive tissue samples

Negative Controls:

• Cell lysates from cells treated with mTOR inhibitors (rapamycin, torin)

• Dephosphorylated samples (via phosphatase treatment)

• Samples with blocking peptides that correspond to the phosphorylated epitope

• Secondary antibody-only controls for immunostaining applications

• PBS or buffer-only controls for immunohistochemistry applications

Specificity Controls:

• Parallel detection with antibodies against total (non-phosphorylated) RPS6KB1

• Detection of downstream targets of RPS6KB1 such as phosphorylated S6 protein

• Knockdown or knockout cell models of RPS6KB1 to confirm signal specificity

How does phosphorylation at T412 differ functionally from other phosphorylation sites on RPS6KB1?

RPS6KB1 contains multiple phosphorylation sites that regulate its activation and function through a complex hierarchy:

The phosphorylation at T412 is particularly important because:

• It occurs in the autoinhibitory domain and relieves inhibition of the catalytic activity

• It creates a conformational change that exposes other phosphorylation sites

• It acts as a priming event that facilitates subsequent phosphorylation at T389, which is essential for complete activation

Research has shown that phosphorylation at T412 can be maintained through an agonist-dependent autophosphorylation mechanism , whereas T389 phosphorylation is more directly controlled by mTORC1 activity. Additionally, inhibitor studies demonstrate that different compounds can selectively affect phosphorylation at specific sites, suggesting distinct regulatory mechanisms .

What technical considerations should be addressed when using Phospho-RPS6KB1 (T412) antibody for kinase activity profiling?

When employing Phospho-RPS6KB1 (T412) antibodies for kinase activity profiling, researchers should consider several technical aspects:

Sample Preparation:

• Rapid lysis is essential to preserve phosphorylation status

• Inclusion of phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in lysis buffers is critical

• Standardizing protein concentration before analysis is necessary for comparative studies

• Consider subcellular fractionation as phosphorylated RPS6KB1 may have different localizations

Antibody Validation:

• Cross-validation with multiple antibodies targeting the same site is recommended

• Western blot confirmation of specificity before using in complex assays

• Peptide competition assays to confirm epitope specificity

• Testing reactivity across different species if working with non-human models

Quantification Methods:

• Use appropriate normalization controls (total RPS6KB1, housekeeping proteins)

• Consider multiplexed detection systems for simultaneous measurement of multiple phosphorylation sites

• Implement phosphoproteomics approaches for comprehensive pathway analysis

• For in silico Kinome Activity Profiling (iKAP), ensure computational models are trained on relevant datasets

Research by Liu et al. demonstrated that using phospho-specific antibodies allowed them to accurately determine that corynoxine (Cory) significantly decreased RPS6KB1 phosphorylation and kinase activity compared to corynoxine B (Cory B) , highlighting the utility of these antibodies in discriminating between closely related compounds with different biological effects.

How can researchers optimize detection of Phospho-RPS6KB1 (T412) in different experimental systems?

Optimizing detection of Phospho-RPS6KB1 (T412) requires tailoring approaches to specific experimental systems:

Cell Culture Systems:

• Serum starvation (12-24 hours) followed by acute stimulation maximizes phosphorylation signal

• Cell density affects baseline phosphorylation; standardize confluency across experiments

• Different cell types may require adjusted lysis conditions due to varying protein abundance

• Consider transfection of fluorescently-tagged S6K1 for live cell imaging studies

Tissue Samples:

• Flash freezing is critical to preserve phosphorylation status

• Optimize fixation protocols for IHC (10% neutral buffered formalin is commonly used)

• Antigen retrieval methods significantly impact phospho-epitope detection

• Consider section thickness (4-6 μm optimal for most applications)

Western Blotting Optimization:

• Use PVDF membranes for better protein retention and signal

• Optimize transfer conditions for high molecular weight proteins

• BSA-based blocking solutions often perform better than milk for phospho-specific antibodies

• Dilution ranges of 1:500-1:2000 typically work well

Immunofluorescence Refinements:

• Permeabilization conditions affect epitope accessibility

• Test both methanol and detergent-based permeabilization methods

• Signal amplification systems may be needed for low abundance targets

• Consider FRET-FLIM imaging technology for monitoring phosphorylation in living cells

As demonstrated by Jastrzebski et al., FRET-FLIM imaging using a novel S6K1 sensor (SensOR) can provide real-time visualization of S6K1 phosphorylation in living cells, allowing for dynamic studies of mTOR signaling that traditional fixed-cell methods cannot achieve .

What are the known cross-reactivity issues with Phospho-RPS6KB1 (T412) antibodies and how can they be addressed?

Several cross-reactivity issues have been reported with Phospho-RPS6KB1 (T412) antibodies that researchers should be aware of:

Common Cross-Reactivity Issues:

• Cross-reactivity with RPS6KB2 (p70 S6 Kinase β) due to sequence homology

• Potential recognition of similarly phosphorylated motifs in other AGC family kinases

• Inconsistent reactivity across species despite predicted cross-reactivity

• Antibody specificity can vary between batches and manufacturers

Addressing Cross-Reactivity:

• Validation Approaches:

  • Parallel testing with multiple antibodies from different manufacturers

  • Using knockout/knockdown models as negative controls

  • Peptide competition assays with both target and potential cross-reactive peptides

  • Phosphatase treatment of samples to confirm phospho-specificity

• Experimental Design Considerations:

  • Include closely related protein controls (e.g., RPS6KB2) in experiments

  • Implement more specific techniques like immunoprecipitation followed by Western blotting

  • Consider mass spectrometry validation for critical experiments

  • Test antibody specificity in your particular experimental system

• Bioinformatic Approaches:

  • Analyze potential cross-reactive epitopes using sequence alignment tools

  • Consider predicted reactivity information provided by manufacturers

  • Consult phosphorylation databases for similar motifs in other proteins

One specific noted issue is that some Phospho-p70 S6 Kinase (Thr421) antibodies detect endogenous levels of p70 S6 Kinase only when phosphorylated at Thr444, which is historically referenced as Thr421 . This highlights the importance of understanding the nomenclature and potential site-specific variations when interpreting results.

How can researchers effectively use Phospho-RPS6KB1 (T412) antibodies for studying mTOR pathway dynamics?

Phospho-RPS6KB1 (T412) antibodies are valuable tools for investigating mTOR pathway dynamics when used strategically:

Temporal Dynamics Analysis:

• Time-course experiments can reveal the kinetics of RPS6KB1 phosphorylation following stimulation

• Pulse-chase approaches with pathway inhibitors can elucidate the stability of phosphorylation

• Real-time monitoring using FRET-FLIM with S6K1 biosensors provides dynamic visualization

• Correlating T412 phosphorylation with T389 phosphorylation reveals activation sequence

Spatial Distribution Studies:

• Immunofluorescence with subcellular markers can map compartment-specific activation

• Subcellular fractionation followed by Western blotting quantifies distribution between compartments

• Live-cell imaging with fluorescently labeled S6K1 allows tracking of translocation events

• Co-localization with mTOR complex components (raptor) provides insight into complex formation

Pathway Crosstalk Investigations:

• Combinatorial treatment with inhibitors of intersecting pathways (PI3K, MAPK, AMPK)

• Correlation of RPS6KB1 phosphorylation with other pathway markers

• Analysis of feedback mechanisms through simultaneous detection of multiple phosphorylation sites

• Investigation of RPS6KB1 phosphorylation in the context of stress responses or nutrient deprivation

Quantitative Approaches:

• Dose-response studies with mTOR pathway modulators

• Computational modeling using phosphorylation data as inputs

• Parallel analysis of multiple downstream targets (4EBP1, PRAS40, S6)

• Application of in silico Kinome Activity Profiling (iKAP) for comprehensive pathway analysis

Research by Jastrzebski et al. demonstrated that FRET-FLIM imaging can reveal the recruitment and phosphorylation of S6K1 in living cells, providing critical insights into the dynamics and localization of mTOR signaling that cannot be captured with fixed-cell techniques .

What troubleshooting approaches are recommended for inconsistent Phospho-RPS6KB1 (T412) detection?

When facing inconsistent detection of Phospho-RPS6KB1 (T412), researchers should systematically address potential issues:

Sample Preparation Issues:

• Problem: Rapid dephosphorylation during processing
  Solution: Ensure samples are kept cold, use phosphatase inhibitor cocktails, minimize processing time

• Problem: Inadequate cell lysis
  Solution: Optimize lysis buffer composition, consider different detergents, verify complete lysis microscopically

• Problem: Protein degradation
  Solution: Add protease inhibitors, avoid freeze-thaw cycles, use fresh samples when possible

Antibody-Related Issues:

• Problem: Antibody batch variation
  Solution: Test new lots against reference samples, consider pooling consistent batches for long-term studies

• Problem: Non-specific binding
  Solution: Optimize blocking conditions, try different blocking agents (BSA vs. milk), increase washing stringency

• Problem: Insufficient antibody concentration
  Solution: Perform antibody titration experiments, adjust concentration based on signal-to-noise ratio

Technical Considerations:

• Problem: Variable phosphorylation levels in samples
  Solution: Standardize culture conditions, synchronize cells, control stimulation timing precisely

• Problem: Antigen masking in fixed tissues
  Solution: Optimize antigen retrieval methods, test multiple fixation protocols

• Problem: High background in immunostaining
  Solution: Increase antibody dilution, extend blocking time, use more stringent washing

Validation Approaches:

• Problem: Uncertain antibody specificity
  Solution: Include positive controls (insulin-stimulated cells), negative controls (phosphatase-treated samples), and peptide competition assays

• Problem: Difficult interpretation of results
  Solution: Compare phosphorylated vs. total protein ratios, include pathway inhibitor controls

A systematic approach to troubleshooting is particularly important when working with phospho-specific antibodies due to the dynamic nature of phosphorylation events and the technical challenges in preserving and detecting these modifications accurately.

How can Phospho-RPS6KB1 (T412) antibodies be integrated into phosphoproteomics workflows?

Integrating Phospho-RPS6KB1 (T412) antibodies into phosphoproteomics workflows enhances their utility for comprehensive signaling pathway analysis:

Immunoaffinity Enrichment Strategies:

• Using phospho-specific antibodies for immunoprecipitation prior to mass spectrometry analysis

• Creating antibody-coupled beads for pulldown of phosphorylated RPS6KB1 and its interacting partners

• Implementing sequential immunoprecipitation to isolate complexes containing multiple phosphorylated proteins

• Coupling with proximity labeling techniques (BioID, APEX) to identify transient interacting partners

Validation of Mass Spectrometry Data:

• Confirming phosphoproteomics hits with targeted antibody-based detection

• Using Western blotting to validate relative quantification from MS data

• Applying reversed-phase protein arrays for high-throughput validation across multiple samples

• Correlating in silico kinase activity predictions with antibody-detected phosphorylation levels

Multiplex Detection Approaches:

• Implementing multiplexed Western blotting to simultaneously detect multiple phosphorylation sites

• Utilizing bead-based multiplex assays for quantitative analysis of pathway components

• Employing sequential probing strategies to analyze multiple modifications on the same blot

• Developing custom antibody panels for pathway-focused analysis

Integration with Functional Genomics:

• Correlating phosphorylation status with transcriptomic changes

• Using CRISPR screens to identify regulators of RPS6KB1 phosphorylation

• Combining with genetic models (knockouts, mutations at specific phosphorylation sites)

• Implementing systems biology approaches to map pathway networks

The work by Liu et al. demonstrates the value of integrating antibody-based detection with computational approaches like in silico Kinome Activity Profiling (iKAP) to comprehensively analyze phosphoproteomics data and accurately predict kinase activities, including RPS6KB1 .

What is the relationship between phosphorylation at T412 and other regulatory modifications of RPS6KB1?

The functional activity of RPS6KB1 is regulated by a complex interplay of multiple phosphorylation sites and other post-translational modifications:

Hierarchical Phosphorylation Regulation:

Cross-regulation with Other Modifications:

• Phosphorylation at S424 by JNK1 occurs in coordination with T412 phosphorylation and affects protein stability

• The absence of IKK2 activation can lead to JNK1-mediated phosphorylation of S6K at T412/S424, resulting in protein destabilization

• Dephosphorylation by PPP1CC (Protein Phosphatase 1) at T412 occurs in mitochondria, suggesting compartment-specific regulation

• Ubiquitination may be influenced by phosphorylation status, affecting protein turnover rates

Functional Consequences:

• The sequence of phosphorylation events (T412→T389→T229) creates a regulated activation cascade

• Different phosphorylation patterns may direct RPS6KB1 to specific subcellular locations

• Phosphorylation at T412/S424 without subsequent T389 phosphorylation can result in a partially active or alternatively regulated enzyme

• Some phosphorylation events may create docking sites for interacting proteins

Research by He et al. demonstrated that JNK1 can phosphorylate S6K, and this phosphorylation makes S6K protein unstable in the absence of IKK2 activation , revealing a complex interplay between different signaling pathways in regulating RPS6KB1 stability and activity.

What methodological considerations are important when studying RPS6KB1 (T412) phosphorylation in disease models?

Researching RPS6KB1 (T412) phosphorylation in disease models requires specific methodological considerations:

Cancer Research Applications:

• Compare phosphorylation levels between tumor and adjacent normal tissues

• Correlate with clinical parameters and patient outcomes

• Consider heterogeneity within tumor samples through single-cell or spatial analysis

• Use phospho-RPS6KB1 (T412) as a potential biomarker for mTOR pathway activation

Metabolic Disease Models:

• Assess tissue-specific phosphorylation patterns (liver, muscle, adipose tissue)

• Monitor changes in response to nutritional status and metabolic challenges

• Correlate with insulin sensitivity and glucose metabolism parameters

• Consider circadian variation in signaling pathway activity

Neurodegenerative Disease Studies:

• Optimize tissue fixation and processing to preserve phospho-epitopes in brain tissue

• Use regionalized analysis to capture brain area-specific differences

• Correlate with protein synthesis rates and synaptic plasticity measures

• Consider age-dependent changes in mTOR pathway regulation

Translational Research Considerations:

• Standardize sample collection and processing protocols for clinical specimens

• Develop validated assays for potential diagnostic applications

• Correlate phosphorylation status with response to targeted therapies

• Implement multiplexed analysis to capture pathway activation comprehensively

Technical Adaptations:

• For fixed tissue analysis: optimize antigen retrieval methods specific to the tissue type

• For limited sample quantities: develop micro-scale Western blotting or single-cell analysis methods

• For longitudinal studies: establish reliable normalization strategies to compare across time points

• For drug development: create high-throughput phospho-detection assays suitable for screening

The application of phospho-specific antibodies in breast carcinoma tissue immunohistochemistry, as demonstrated in multiple studies , highlights the utility of these tools in translational research connecting basic mechanisms to clinical observations.

How do different experimental conditions affect the detection and interpretation of RPS6KB1 T412 phosphorylation?

The detection and interpretation of RPS6KB1 T412 phosphorylation are significantly influenced by experimental conditions:

Cell Culture Variables:

• Serum Conditions: Serum starvation (12-24h) followed by acute stimulation maximizes dynamic range of phosphorylation

• Cell Density: Confluence affects basal phosphorylation; standardize across experiments

• Passage Number: Signaling characteristics can drift with extended passage; use cells within defined passage range

• Media Formulation: Amino acid and glucose concentration affect mTOR pathway activation

Stimulation Parameters:

• Duration: Rapid (5-30 min) stimulation with insulin or growth factors induces robust phosphorylation

• Concentration: Dose-response relationships should be established (e.g., insulin 0.01-1 U/ml)

• Pre-treatments: Prior exposure to inhibitors or stressors alters responsiveness

• Combined Stimuli: Multiple simultaneous pathway activators can show synergistic or antagonistic effects

Sample Processing Factors:

• Lysis Conditions: Buffer composition critically affects phospho-epitope preservation

• Temperature Control: Cold processing essential to prevent phosphatase activity

• Timing: Minimize delay between stimulation and lysis/fixation

• Fixation Methods: For tissues/cells, fixative choice and duration affect epitope accessibility

Analytical Considerations:

• Antibody Dilution: Optimal ranges differ by application (WB: 1:500-1:2000; IHC: 1:50-1:300)

• Detection System: Enhanced chemiluminescence vs. fluorescent detection affects sensitivity and dynamic range

• Normalization Strategy: Normalizing to total RPS6KB1 vs. housekeeping proteins yields different insights

• Quantification Method: Densitometry techniques influence the measured phosphorylation levels

Research by He et al. demonstrated that kinase assay conditions, including buffer composition (HEPES, MgCl2, glycerophosphate, DTT, ATP concentrations) and incubation parameters (37°C for 30 min), significantly impact the ability to detect phosphorylation events accurately , underlining the importance of standardized conditions for reproducible results.

What are the current best practices for validating Phospho-RPS6KB1 (T412) antibody specificity?

Establishing confident antibody specificity is crucial for reliable research. Current best practices for validating Phospho-RPS6KB1 (T412) antibodies include:

Primary Validation Approaches:

• Phosphatase Treatment: Treating one sample portion with lambda phosphatase should eliminate signal from phospho-specific antibodies

• Peptide Competition: Pre-incubation with phosphorylated and non-phosphorylated peptides should selectively block specific binding

• Genetic Models: Using CRISPR-Cas9 knockout or knockdown models of RPS6KB1 to confirm signal specificity

• Stimulation/Inhibition: Treatment with known pathway activators (insulin) and inhibitors (rapamycin) should show expected modulation of signal

Advanced Validation Methods:

• Phospho-site Mutants: Testing antibody reactivity against T412A mutant RPS6KB1 should eliminate specific signal

• Mass Spectrometry Confirmation: MS analysis of immunoprecipitated proteins confirms presence of the phosphorylated epitope

• Cross-platform Confirmation: Correlating results across different detection methods (WB, ELISA, IHC)

• Multi-antibody Concordance: Comparing results using antibodies from different vendors or clones targeting the same site

Documentation and Reporting Standards:

• Complete Antibody Information: Report catalog number, lot, clonality, and host species

• Validation Evidence: Include validation controls in publications

• Protocol Transparency: Provide detailed methods including blocking agents, dilutions, and incubation conditions

• Imaging Parameters: Report exposure times, gain settings, and image processing steps

Emerging Best Practices:

• Recombinant Antibody Standards: Using sequenced recombinant antibodies improves reproducibility

• Multi-site Testing: Validating across different laboratories and experimental systems

• Application-specific Validation: Confirming specificity for each application (WB vs. IHC) independently

• Batch Testing: Validating new lots against reference standards