Phospho-RPS6KB1 (Ser371) Antibody

What is Phospho-RPS6KB1 (Ser371) and why is it significant in cellular signaling?

Phospho-RPS6KB1 (Ser371) refers to the ribosomal protein S6 kinase beta-1 when it is phosphorylated at the serine residue at position 371. RPS6KB1 functions as a downstream effector of mTOR (mammalian target of rapamycin) signaling, acting in response to growth factors and nutrients to promote cell proliferation, cell growth, and cell cycle progression . The phosphorylation at Ser371 represents one of several critical post-translational modifications that regulate RPS6KB1 activity.

RPS6KB1 plays a central role in protein synthesis by phosphorylating multiple substrates including EIF4B, RPS6, and EEF2K . Its activity contributes significantly to cell survival mechanisms by repressing pro-apoptotic functions. The phosphorylation state at Ser371 specifically serves as a regulatory mechanism that helps determine the kinase's activity level and substrate specificity.

Research has shown that RPS6KB1 is not merely overexpressed in certain cancers but is often hyperphosphorylated, suggesting that its activation state rather than just protein abundance may be crucial in pathological processes . This distinction highlights why studying specific phosphorylation sites like Ser371 is particularly important for understanding disease mechanisms.

How does phosphorylation at Ser371 interact with other phosphorylation sites on RPS6KB1?

RPS6KB1 activation involves a complex series of phosphorylation events at multiple sites. Phosphorylation at Ser371 occurs within the context of a coordinated phosphorylation cascade that includes other critical sites such as Thr389, Thr229, Thr421, and Ser424 . These phosphorylation events work in concert to fully activate the kinase and direct its substrate specificity.

Interestingly, recent research indicates that multisite phosphorylation patterns on RPS6KB1 can actually direct its substrate specificity. For example, while phosphorylation at the canonical activation sites (Thr229, Ser371, and Thr389) enables standard RPS6KB1 function, additional phosphorylation at sites in the C-terminal domain (CTD) like Ser424 can redirect the kinase toward alternative substrates . This "phospho-code" represents an elegant regulatory mechanism that allows the same kinase to serve multiple signaling pathways depending on its specific phosphorylation pattern.

What experimental applications are most suitable for Phospho-RPS6KB1 (Ser371) Antibody?

Phospho-RPS6KB1 (Ser371) antibodies are versatile tools that can be employed in multiple experimental applications. Based on the product specifications, these antibodies are particularly suitable for:

• Western Blot (WB): The primary application for detecting phosphorylation status of RPS6KB1 at Ser371 in cell or tissue lysates. Typical dilution ranges for this application are 1:500-1:2000 .

• Immunohistochemistry (IHC): For visualizing the spatial distribution of phosphorylated RPS6KB1 in tissue sections, using dilutions of approximately 1:100-1:300 .

• Immunofluorescence (IF): For cellular localization studies of phosphorylated RPS6KB1, typically at dilutions of 1:50-1:200 .

• ELISA: For quantitative assessment of phosphorylated RPS6KB1 levels, often using higher dilutions around 1:20000 .

These applications enable researchers to investigate the phosphorylation state of RPS6KB1 at Ser371 across different experimental contexts, from protein expression levels to cellular localization. When selecting an antibody for specific applications, researchers should consider factors such as species reactivity (human, mouse, rat are common), clonality (polyclonal versus monoclonal), and validation data for the specific application.

The ability to detect phospho-specific modifications provides critical information about activation states in signaling cascades that would be impossible to obtain with antibodies recognizing only the total protein.

What are the best practices for using Phospho-RPS6KB1 (Ser371) Antibody in Western Blot experiments?

Western blotting with phospho-specific antibodies requires particular attention to sample preparation and experimental conditions to preserve phosphorylation states and ensure specificity. For optimal results with Phospho-RPS6KB1 (Ser371) antibody:

Sample Preparation:

• Always include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in your lysis buffer to prevent dephosphorylation during extraction.

• Process samples quickly and maintain cold temperatures throughout to minimize phosphatase activity.

• Consider using stimulated cells (e.g., following growth factor treatment) as positive controls, as RPS6KB1 phosphorylation is regulated by growth factors and nutrients .

Protocol Optimization:

• Use recommended dilutions (1:500-1:2000) but optimize for your specific experimental conditions .

• Include both positive controls (lysates from cells treated with growth factors) and negative controls (lysates from cells treated with mTOR inhibitors like rapamycin).

• Consider including a phosphatase-treated sample as an additional negative control to confirm antibody specificity.

Interpretation:

• Always run parallel blots for total RPS6KB1 to normalize phosphorylation signals.

• When quantifying results, express data as the ratio of phosphorylated to total protein.

• Confirm band specificity by molecular weight (approximately 70 kDa for RPS6KB1).

The most common issue with phospho-specific antibodies is non-specific binding. Thorough blocking (5% BSA in TBST is often preferred over milk for phospho-antibodies) and proper washing steps are essential for reducing background. If detecting endogenous levels of phosphorylated protein, consider signal enhancement methods or highly sensitive detection systems due to potentially low abundance of the phosphorylated form.

How can Phospho-RPS6KB1 (Ser371) Antibody be effectively used in immunohistochemistry applications?

Immunohistochemistry (IHC) with phospho-specific antibodies presents unique challenges but can provide valuable spatial information about kinase activation states in tissues. For optimal IHC with Phospho-RPS6KB1 (Ser371) antibody:

Tissue Preparation and Fixation:

• Rapid fixation is critical for preserving phosphorylation states; delayed fixation can result in significant loss of phospho-epitopes.

• Formalin-fixed paraffin-embedded (FFPE) tissues require proper antigen retrieval, typically heat-induced in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

• Fresh-frozen sections may better preserve phospho-epitopes but require careful handling to maintain tissue morphology.

Protocol Considerations:

• Use recommended dilutions (1:100-1:300) as a starting point for optimization .

• Extended primary antibody incubation (overnight at 4°C) often yields better results than shorter incubations.

• Signal amplification systems like tyramide signal amplification (TSA) may be necessary for detecting low-abundance phospho-proteins.

• Always include appropriate positive and negative controls, including phosphatase-treated sections.

Counterstaining and Visualization:

• Hematoxylin counterstaining should be light to avoid masking specific signals.

• For fluorescent detection, DAPI nuclear counterstain works well.

• Consider using multispectral imaging or confocal microscopy for co-localization studies.

Importantly, validation of phospho-specific staining can be achieved by treating adjacent tissue sections with lambda phosphatase prior to immunostaining. This should dramatically reduce or eliminate specific staining while leaving non-specific background unchanged. Additionally, comparing staining patterns in normal versus stimulated tissues or comparing phospho-RPS6KB1 staining with downstream effector phosphorylation can provide functional validation of the observed patterns.

What controls should be included when validating experimental results with Phospho-RPS6KB1 (Ser371) Antibody?

Proper controls are crucial for validating experiments with phospho-specific antibodies. For Phospho-RPS6KB1 (Ser371) antibody, consider the following comprehensive control strategy:

Positive Controls:

• Lysates from cells treated with growth factors known to activate the mTOR pathway (e.g., insulin, IGF-1, EGF).

• Tissues with known high expression of activated RPS6KB1 (e.g., certain tumor types).

• Recombinant phosphorylated RPS6KB1 protein (if available).

Negative Controls:

• Lysates from cells treated with mTOR inhibitors (e.g., rapamycin, Torin1) or PI3K inhibitors (e.g., LY294002).

• Lysates from cells in which RPS6KB1 has been knocked down using siRNA or CRISPR/Cas9.

• Phosphatase-treated samples (lambda phosphatase treatment eliminates phospho-epitopes).

Specificity Controls:

• Peptide competition assay using the phospho-peptide used as immunogen to confirm binding specificity.

• Parallel staining/blotting with antibodies recognizing different phosphorylation sites on RPS6KB1.

• Comparison with an antibody recognizing total RPS6KB1 regardless of phosphorylation state.

Functional Validation:

• Correlation of Ser371 phosphorylation with phosphorylation of known RPS6KB1 substrates like RPS6.

• Pharmacological manipulation using specific inhibitors like LY2584702, which has been shown to inhibit RPS6KB1 phosphorylation in lung adenocarcinoma cell lines .

These controls should be systematically implemented across different experimental platforms. For example, in cell-based studies, researcher may examine how phosphorylation changes upon serum starvation and refeeding, or in response to specific growth factors or inhibitors. In tissue-based studies, comparing normal and pathological samples can provide important context for interpreting phosphorylation signals.

How does the phospho-code of RPS6KB1 direct substrate specificity and signaling outcomes?

Recent research has revealed that RPS6KB1 employs a sophisticated "phospho-code" where combinations of phosphorylation events across multiple sites directly influence substrate selection. This code extends beyond simple binary activation to create functionally distinct kinase variants that target different substrates.

The C-terminal domain (CTD) of RPS6KB1 contains a cluster of phospho-sites bearing the Ser/Thr-Pro motif which significantly contribute to determining substrate specificity . Experimental evidence demonstrates this principle clearly: deletion of the CTD from S6K1 specifically decreased phosphorylation of EPRS (a non-canonical substrate) while having minimal impact on phosphorylation of RPS6 (the canonical substrate) .

What makes this system particularly sophisticated is the involvement of multiple kinases in establishing the phospho-code. While mTORC1 phosphorylates the signature site Thr389, other kinases like Cdk5 can phosphorylate specific sites in the CTD, particularly Ser424 and Ser429 . This creates a "proteoform" of S6K1 (termed S6K1*) that exhibits altered substrate preference.

The phospho-code concept helps explain previously puzzling observations:

This emerging understanding of the RPS6KB1 phospho-code suggests that researchers should consider not only whether RPS6KB1 is activated but which specific "version" of activated RPS6KB1 is present in their experimental system. Different phosphorylation patterns may indicate engagement of distinct downstream pathways, adding a layer of complexity to mTOR signaling that was previously underappreciated.

What is the significance of RPS6KB1 phosphorylation at Ser371 in cancer research?

The phosphorylation state of RPS6KB1, rather than merely its expression level, appears to play a critical role in cancer progression and potential therapeutic targeting. Research has revealed several important aspects of phosphorylated RPS6KB1 in cancer:

Hyperphosphorylation in Cancer:
Studies in non-small cell lung cancer (NSCLC) have demonstrated that RPS6KB1 is over-activated through phosphorylation rather than just overexpressed at the protein level . This suggests that measuring phospho-RPS6KB1 may provide more clinically relevant information than assessing total protein levels.

Prognostic Value:
Phosphorylated RPS6KB1 has shown independent prognostic value in multivariate Cox regression analysis of cancer patients, potentially serving as a biomarker for disease progression and patient outcomes .

Therapeutic Targeting:
Specific inhibition of RPS6KB1 phosphorylation using compounds like LY2584702 has demonstrated significant effects on cancer cell behavior, including:

• Suppressed cell proliferation

• Increased G0-G1 phase cell cycle arrest

• Enhanced apoptosis, particularly in lung adenocarcinoma cell lines

These findings have important implications for experimental design in cancer research. When investigating mTOR/S6K1 signaling in cancer models, researchers should consider:

• Measuring multiple phosphorylation sites on RPS6KB1 (including Ser371) rather than focusing solely on Thr389

• Correlating phosphorylation patterns with specific functional outcomes

• Examining how different cancer therapies affect the pattern of RPS6KB1 phosphorylation

• Exploring combination approaches that target both mTOR and other kinases that contribute to the RPS6KB1 phospho-code

The observation that dephosphorylation of RPS6KB1 can promote apoptosis in cancer cells suggests that phospho-RPS6KB1 antibodies may be valuable tools not only for basic research but also for drug discovery efforts aimed at developing novel cancer therapeutics that modulate specific phosphorylation events.

How can researchers distinguish between different phosphorylated forms of RPS6KB1 in complex cellular contexts?

Distinguishing between different phosphorylated forms of RPS6KB1 in complex cellular environments requires sophisticated experimental approaches. Here are methodological strategies for comprehensive phosphorylation profiling:

Multiplexed Phospho-Antibody Analysis:

• Sequential probing with site-specific phospho-antibodies (Ser371, Thr389, Thr421, etc.)

• Multiplex fluorescent Western blotting with spectrally distinct secondary antibodies

• Phospho-flow cytometry for single-cell analysis of multiple phosphorylation sites

Mass Spectrometry-Based Approaches:

• Phospho-peptide enrichment followed by LC-MS/MS analysis

• Parallel reaction monitoring (PRM) for targeted quantification of specific phospho-sites

• SILAC or TMT labeling for comparative phosphoproteomics across conditions

Proximity-Based Detection Methods:

• Proximity ligation assay (PLA) to detect co-occurrence of multiple phospho-sites

• FRET-based biosensors designed to detect specific phosphorylation patterns

Functional Readouts:

• Substrate-specific kinase assays using known substrates of differently phosphorylated RPS6KB1

• Correlation of phosphorylation patterns with downstream signaling events

Experimental Design Considerations:

When designing experiments to distinguish different phosphorylated forms, it's crucial to include appropriate stimulation conditions. For example, rapamycin treatment will inhibit mTORC1-dependent phosphorylation at Thr389 while potentially leaving other sites intact. Similarly, Cdk5 inhibitors might specifically affect CTD phosphorylation sites like Ser424 and Ser429 without altering canonical activation sites .

The evidence that different phosphorylation patterns direct RPS6KB1 toward different substrates provides a functional readout: researchers can monitor phosphorylation of canonical substrates (RPS6) versus non-canonical substrates (EPRS) as an indicator of which "version" of phosphorylated RPS6KB1 predominates in their experimental system.

Why might Phospho-RPS6KB1 (Ser371) Antibody show inconsistent results across different experimental conditions?

Inconsistent results with phospho-specific antibodies like Phospho-RPS6KB1 (Ser371) can stem from multiple sources. Understanding these variables is crucial for experimental reproducibility:

Sample Preparation Issues:

• Inadequate phosphatase inhibition during lysis can lead to rapid dephosphorylation

• Variations in sample handling time and temperature affect phosphorylation stability

• Different lysis buffers may extract phosphorylated proteins with varying efficiency

• Freeze-thaw cycles can degrade phospho-epitopes over time

Biological Variables:

• Cell culture conditions (confluence, passage number, serum batch) affect baseline phosphorylation

• Timing of stimulation/inhibition treatments influences phosphorylation dynamics

• Metabolic state of cells/tissues impacts mTOR pathway activity

• Heterogeneity within tissue samples creates variable phosphorylation profiles

Technical Considerations:

• Antibody batch-to-batch variations affect epitope recognition

• Storage conditions and repeated freeze-thaw of antibody can reduce activity

• Buffer composition (primary antibody diluent, blocking buffer) influences specificity

• Detection system sensitivity may be insufficient for low-abundance phospho-proteins

Protocol Optimization Strategies:

When troubleshooting inconsistent results, a systematic approach is essential. Begin by standardizing sample preparation and handling across experiments. Then evaluate antibody performance using well-characterized positive and negative controls. Finally, optimize detection conditions to ensure both sensitivity and specificity.

It's worth noting that some phosphorylation events are inherently transient and may show natural variation even under seemingly identical conditions. Time-course experiments can help characterize the phosphorylation dynamics and identify optimal timepoints for consistent detection.

How can specificity of Phospho-RPS6KB1 (Ser371) Antibody be rigorously validated?

Rigorous validation of phospho-specific antibodies is essential for generating reliable data. For Phospho-RPS6KB1 (Ser371) antibody, a comprehensive validation strategy should include:

Biochemical Validation:

• Peptide competition assays: Pre-incubating the antibody with the phospho-peptide immunogen should abolish specific signal

• Phosphatase treatment: Treating samples with lambda phosphatase should eliminate signal from phospho-specific antibodies

• Dot blot analysis: Testing antibody against phosphorylated and non-phosphorylated peptides to confirm specificity

Genetic Validation:

• Mutant expression: Testing antibody reactivity against wild-type RPS6KB1 versus S371A mutant (cannot be phosphorylated)

• Knockout/knockdown controls: Confirming absence of signal in RPS6KB1-depleted samples

• Overexpression systems: Comparing endogenous versus overexpressed protein detection

Pharmacological Validation:

• Kinase inhibitors: Confirming reduced signal after treatment with relevant pathway inhibitors

• Stimulation experiments: Demonstrating increased signal following pathway activation

• Dose-response relationships: Showing graded responses to inhibitors/activators

Cross-Platform Validation:

• Orthogonal techniques: Confirming phosphorylation using mass spectrometry

• Multiple applications: Validating specificity across Western blot, IHC, IF, etc.

• Reproducibility: Demonstrating consistent results across different lots and laboratories

For Phospho-RPS6KB1 (Ser371) specifically, validation should include experiments leveraging the known biology of this phosphorylation site. The fact that Ser371 phosphorylation is maintained by an agonist-dependent autophosphorylation mechanism suggests that mTOR inhibitors should reduce this phosphorylation, providing a pharmacological validation approach.

Additionally, the specificity statement from commercial antibodies can guide validation: "Phospho-p70 S6 kinase Alpha (S371) Polyclonal Antibody detects endogenous levels of p70 S6 kinase Alpha protein only when phosphorylated at S371" . This claim should be rigorously tested through the approaches outlined above.

What methodological considerations are essential when comparing results across different phospho-specific RPS6KB1 antibodies?

When comparing results obtained with different phospho-specific antibodies targeting various sites on RPS6KB1, researchers must consider several methodological factors to ensure valid comparisons:

Antibody Characteristics:

• Clonality differences: Polyclonal antibodies may recognize multiple epitopes around the phosphorylation site, while monoclonals recognize a single epitope

• Host species variations: Different host species (rabbit vs. mouse) may affect background patterns and secondary antibody compatibility

• Epitope coverage: The exact peptide sequence used as immunogen influences antibody specificity and sensitivity

Protocol Standardization:

• Identical sample preparation: Use the same lysis buffers, phosphatase inhibitors, and handling procedures

• Consistent blocking conditions: Standardize blocking agents (BSA vs. milk) and concentrations

• Equivalent antibody concentrations: Adjust dilutions based on antibody concentration (typically 1 mg/mL)

• Matched incubation conditions: Use identical temperature, time, and buffer compositions

Comparative Analysis Approach:

Physiological Context Considerations:

• Different phosphorylation sites may have distinct kinetics of phosphorylation/dephosphorylation

• Some sites may be more sensitive to specific stimuli than others

• Phosphorylation events may have interdependencies (hierarchical phosphorylation)

For example, when comparing antibodies targeting phospho-Ser371 versus phospho-Thr389, researchers should recognize that these sites are regulated differently: Thr389 is directly phosphorylated by mTORC1, while Ser371 involves an autophosphorylation mechanism . This means that the two sites may respond with different kinetics and sensitivities to mTOR pathway modulators.

To ensure meaningful comparisons, time-course experiments following stimulation or inhibition can help characterize the phosphorylation dynamics at each site. Additionally, dose-response studies can reveal differential sensitivities of various phosphorylation sites to the same stimulus.