Phospho-RPS6KA1 (S380) Recombinant Monoclonal Antibody

What is RPS6KA1 and why is its phosphorylation at S380 significant?

RPS6KA1 (Ribosomal Protein S6 Kinase Alpha-1), also known as RSK1, is a serine/threonine-protein kinase that functions downstream of the ERK (MAPK1/ERK2 and MAPK3/ERK1) signaling pathway. It plays crucial roles in mediating mitogenic and stress-induced activation of transcription factors including CREB1, ETV1/ER81, and NR4A1/NUR77 . The S380 site is particularly important because its phosphorylation is a critical regulatory mechanism that enables cells to respond to extracellular signals and stressors, thereby modulating gene expression and influencing various cellular processes including cell growth and stress responses . Dysregulation of S380 phosphorylation can significantly impact diseases related to cell proliferation and aberrant gene expression.

How does RPS6KA1 contribute to cellular signaling networks?

RPS6KA1 serves as a multifunctional node in cellular signaling networks by:

• Regulating translation through phosphorylation of RPS6 and EIF4B

• Mediating cellular proliferation, survival, and differentiation by modulating mTOR signaling

• Repressing pro-apoptotic functions of BAD and DAPK1

• Facilitating EGF-stimulated phosphorylation of CREB1 in fibroblasts, leading to transcriptional activation of immediate-early genes

• Indirectly regulating transcription of multiple genes by phosphorylating GSK3B at Ser-9 upon insulin stimulation

This extensive involvement in multiple pathways makes RPS6KA1 phosphorylation status a critical indicator of several cellular processes and potential therapeutic target.

What distinguishes phosphorylation of RPS6 via RPS6KA1 (ERK pathway) from phosphorylation via RPS6KB1 (mTOR pathway)?

The phosphorylation of RPS6 can occur through two distinct pathways with different biological implications:

Recent research using Phospho-seq demonstrated that chromatin accessibility at the RPS6KA1 gene correlates with pRPS6 levels, while accessibility at RPS6KB1 does not show the same correlation, suggesting that in certain contexts (e.g., diencephalic lineage), ERK signaling through RPS6KA1 may play a more dominant role in RPS6 phosphorylation than mTOR signaling .

What are the optimal protocols for using Phospho-RPS6KA1 (S380) antibodies in Western Blot experiments?

For optimal Western Blot results with Phospho-RPS6KA1 (S380) antibodies, researchers should follow these methodological guidelines:

• Sample preparation: Prepare cell lysates from appropriate experimental conditions (e.g., stimulated with EGF, PMA, or PMA+Ionomycin for 20 minutes to induce phosphorylation)

• Antibody dilution: Use at 1:500-1:5000 dilution range, with 1:1000 being a common starting point

• Detection system: Utilize HRP-conjugated secondary antibodies against rabbit IgG (as the primary antibody is typically rabbit-derived)

• Expected results: Look for a specific band at approximately 90-93 kDa under reducing conditions

• Controls: Include both positive controls (cells treated with pathway activators) and negative controls (untreated cells or pathway inhibitors) to verify antibody specificity

• Buffer conditions: Use Immunoblot Buffer Group 1 or equivalent for optimal signal-to-noise ratio

When troubleshooting, consider that phospho-specific antibodies often require more stringent blocking conditions and freshly prepared lysates to prevent dephosphorylation by endogenous phosphatases.

How can Phospho-RPS6KA1 (S380) antibodies be effectively utilized in immunoprecipitation experiments?

For successful immunoprecipitation (IP) with Phospho-RPS6KA1 (S380) antibodies:

• Antibody amount: Use 3μg of antibody per 1mg of whole cell lysate

• Recommended dilution: Follow a 1:200-1:1000 dilution range, optimizing based on experimental conditions

• IP protocol:

  • Bind antibody to appropriate protein A/G beads

  • Incubate with cell lysate (preferably freshly prepared)

  • Wash thoroughly to remove non-specific interactions

  • Elute bound protein complexes for downstream analysis

• Detection method: For western blotting of immunoprecipitated samples, use HRP-conjugated Protein G antibody as the secondary antibody (1/2000 dilution)

• Controls: Include a rabbit control IgG immunoprecipitation to identify non-specific binding

Experimental data shows that when immunoprecipitating from HeLa whole cell lysate, a clear band at 90 KDa can be observed in the Phospho-RPS6KA1 (S380) antibody lane compared to the control IgG lane, confirming specificity .

What considerations are important for immunocytochemistry applications of Phospho-RPS6KA1 (S380) antibodies?

When performing immunocytochemistry with Phospho-RPS6KA1 (S380) antibodies:

• Cell preparation: Use immersion fixation for cell lines like HeLa, with and without stimulation (e.g., PMA) to observe differential phosphorylation

• Antibody concentration: Apply at approximately 25 μg/mL and incubate for 3 hours at room temperature

• Detection system: Use fluorescently-conjugated secondary antibodies (e.g., NorthernLights™ 557-conjugated Anti-Rabbit IgG)

• Counterstaining: Include nuclear counterstain (DAPI) to localize cellular compartments

• Expected localization: Specific staining should be observed in both plasma membranes and cytoplasm, with differences in intensity between stimulated and unstimulated cells

• Signal validation: Compare staining patterns with Western blot results from the same experimental conditions to confirm specificity

This approach allows visualization of both the abundance and subcellular localization of phosphorylated RPS6KA1, providing insights not obtainable through biochemical methods alone.

How can Phospho-RPS6KA1 (S380) antibodies be integrated with Phospho-seq technology?

Phospho-seq represents an advanced integrated approach that aims to quantify cytoplasmic and nuclear proteins, including those with post-translational modifications such as phosphorylation . To integrate Phospho-RPS6KA1 (S380) antibodies with Phospho-seq:

• Antibody validation: First validate the antibody's specificity in conventional assays (WB, IP) before inclusion in Phospho-seq panels

• Panel design: Include Phospho-RPS6KA1 (S380) antibodies alongside antibodies against relevant pathway components (e.g., total RPS6KA1, downstream targets, or parallel pathway components)

• Experimental design:

  • Include appropriate cellular treatments (e.g., pathway activators like EGF for 1h or inhibitors like PX-866 for 4h)

  • Use cell surface hashing antibodies for multiplexed profiling of cells in resting, activated, or inhibited conditions

• Data interpretation: Correlate phosphorylation signals with:

  • Chromatin accessibility data from scATAC-seq profiles

  • Expression of pathway components

  • Cellular phenotypes or states

This approach allows researchers to simultaneously observe phosphorylation in the context of other cellular parameters, providing a more comprehensive view of signaling dynamics across different cell states or types.

What are effective strategies to validate the specificity of Phospho-RPS6KA1 (S380) antibodies?

Rigorous validation of phospho-specific antibodies is essential for reliable research findings. For Phospho-RPS6KA1 (S380) antibodies, consider these methodological approaches:

• Pathway modulation:

  • Positive control: Treat cells with activators like PMA, EGF, or PMA+Ionomycin to increase phosphorylation

  • Negative control: Use pathway inhibitors or phosphatase treatment to reduce phosphorylation

• Multiple detection methods: Confirm specificity across different techniques:

  • Western blot (observe band at expected 90-93 kDa)

  • Immunoprecipitation followed by mass spectrometry

  • Immunocytochemistry (observe expected subcellular localization)

• Genetic validation:

  • Use RPS6KA1 knockout or knockdown systems

  • Employ site-directed mutagenesis (S380A) to create a non-phosphorylatable control

• Cross-reactivity assessment:

  • Test against related kinases (other RSK family members)

  • Evaluate specificity across species (human, mouse, rat) if relevant

• Peptide competition assay:

  • Pre-incubate antibody with phospho-peptide immunogen

  • Signal should be blocked by specific phospho-peptide but not by non-phosphorylated equivalent

These validation strategies ensure that experimental observations truly reflect the phosphorylation state of RPS6KA1 rather than artifacts or cross-reactivity.

How can researchers distinguish between activity-dependent phosphorylation and constitutive phosphorylation of RPS6KA1?

Distinguishing between activity-dependent and constitutive phosphorylation requires systematic experimental design:

• Temporal dynamics analysis:

  • Perform time-course experiments (minutes to hours) after stimulation

  • Monitor phosphorylation levels at S380 alongside other phosphorylation sites on RPS6KA1

  • Map the kinetics of phosphorylation appearance and disappearance

• Pathway inhibitor studies:

  • Use specific inhibitors targeting upstream kinases (MEK inhibitors for ERK pathway)

  • Apply combinations of inhibitors to identify convergent pathways

  • Quantify the extent of phosphorylation reduction under different inhibition conditions

• Phosphorylation site comparison:

  • Compare phosphorylation at S380 with other RPS6KA1 phosphorylation sites (T359+S363)

  • Different sites may show different kinetics or responses to stimuli

• Single-cell analysis:

  • Use Phospho-seq or other single-cell technologies to assess heterogeneity in phosphorylation

  • Correlate with cell cycle stage or other cellular states

  • Identify subpopulations with different baseline phosphorylation levels

This multilayered approach can reveal whether S380 phosphorylation is primarily a regulatory mechanism responding to specific signals or maintains baseline activity of RPS6KA1.

How should researchers interpret changes in RPS6KA1 phosphorylation across different cell types and experimental conditions?

Interpretation of RPS6KA1 phosphorylation data requires careful consideration of biological context:

Researchers should avoid simple binary interpretations (phosphorylated vs. non-phosphorylated) and instead focus on the quantitative aspects and biological context of phosphorylation changes.

What experimental controls are essential when studying RPS6KA1 phosphorylation in relation to the ERK and mTOR signaling pathways?

A robust experimental design for studying RPS6KA1 phosphorylation should include these essential controls:

• Pathway-specific positive controls:

  • ERK pathway: EGF or PMA stimulation (20 minutes)

  • mTOR pathway: Insulin stimulation

  • Dual pathway: Serum stimulation after starvation

• Pathway-specific inhibitor controls:

  • ERK pathway: MEK inhibitors (U0126, PD98059)

  • mTOR pathway: Rapamycin or PX-866

  • Combination treatments to assess pathway crosstalk

• Total protein controls:

  • Total RPS6KA1 antibody in parallel with phospho-specific antibody

  • Total RPS6 alongside phospho-RPS6

• Loading and technical controls:

  • Housekeeping proteins (β-actin, GAPDH)

  • Phosphorylation-independent proteins in the same pathway

• Cell type reference controls:

  • Cell lines with known pathway activation status (e.g., K562 vs. iPSCs)

  • Positive and negative cell types for immunohistochemistry

By incorporating these controls, researchers can confidently attribute observed phosphorylation changes to specific pathway activities and distinguish between RPS6KA1-mediated and RPS6KB1-mediated phosphorylation events.

How can researchers troubleshoot inconsistent results when using Phospho-RPS6KA1 (S380) antibodies?

When encountering inconsistent results with Phospho-RPS6KA1 (S380) antibodies, consider these methodological troubleshooting approaches:

• Sample preparation issues:

  • Ensure rapid sample processing to prevent dephosphorylation

  • Include phosphatase inhibitors in lysis buffers

  • Verify protein integrity through total protein staining

  • Avoid repeated freeze-thaw cycles of lysates

• Technical optimization:

  • Test multiple antibody dilutions (1:500-1:5000 for WB, 1:200-1:1000 for IP)

  • Optimize blocking conditions (BSA may be superior to milk for phospho-epitopes)

  • Adjust incubation times and temperatures

  • Consider enhanced detection systems for low-abundance phospho-proteins

• Antibody-specific considerations:

  • Verify antibody lot consistency

  • Test alternative phospho-specific antibodies targeting the same site

  • Determine if the antibody performs better in certain applications (WB vs. IP vs. ICC)

• Experimental design refinement:

  • Adjust stimulation conditions (concentration, duration)

  • Include positive controls with strong phosphorylation signals

  • Consider cell density and culture conditions that may affect signaling

• Quantification approaches:

  • Use quantitative methods like Simple Western

  • Normalize phospho-signal to total protein rather than housekeeping genes

  • Apply appropriate statistical analyses for replicate experiments

Methodical troubleshooting focused on these areas can help identify sources of variability and improve consistency in phosphorylation detection.

How is the study of RPS6KA1 phosphorylation contributing to our understanding of cell type-specific signaling?

Recent research reveals important insights into cell type-specific RPS6KA1 signaling:

• Differential phosphorylation in specialized cell types:

  • Significant differences in pRPS6 levels between photoreceptor subtypes (rods vs. cones) have been observed, reflecting their different metabolic demands

  • These findings suggest that ERK pathway activation through RPS6KA1 may be tailored to cell-specific functions

• Developmental lineage differences:

  • Research using Phospho-seq has identified increased phosphorylation of MAPK/ERK pathway members (including upstream regulators of RPS6KA1) in diencephalic lineages compared to telencephalic lineages

  • This suggests developmental regulation of the pathway activity

• Integration with epigenetic regulation:

  • Chromatin accessibility at the RPS6KA1 gene correlates with pRPS6 levels, indicating a potential epigenetic component to phosphorylation regulation

  • This suggests that cell type-specific chromatin organization may influence RPS6KA1 expression and subsequent phosphorylation activity

• Multi-modal analysis:

  • New techniques like Phospho-seq allow simultaneous measurement of phosphorylation alongside chromatin accessibility and other cellular parameters, enabling more comprehensive understanding of cell-specific signaling networks

These findings highlight the importance of studying phosphorylation within specific cellular contexts rather than in artificial cell line systems alone.

What emerging technologies are enhancing our ability to study RPS6KA1 phosphorylation dynamics?

Cutting-edge technologies are transforming phosphorylation research:

• Integrated multi-modal approaches:

  • Phospho-seq enables simultaneous profiling of phosphorylated proteins, total proteins, and chromatin accessibility in the same cells

  • This integration allows direct correlation between phosphorylation states and gene regulatory elements

• Single-cell phospho-proteomics:

  • New methods allow phosphorylation analysis at single-cell resolution, revealing heterogeneity within seemingly uniform populations

  • This can uncover subpopulations with distinct signaling states that would be masked in bulk analysis

• Live-cell phosphorylation sensors:

  • FRET-based sensors for real-time monitoring of RPS6KA1 phosphorylation

  • Enables temporal dynamics studies in living cells

• CRISPR-based functional genomics:

  • Precise manipulation of phosphorylation sites through base editing

  • Creation of phospho-mimetic or phospho-null mutations to study functional consequences

• Spatial proteomics:

  • Methods like imaging mass cytometry allow visualization of phosphorylation patterns with spatial context

  • Can reveal microenvironmental influences on RPS6KA1 phosphorylation

These technologies are shifting phosphorylation research from static snapshots to dynamic, spatially-resolved understanding of signaling networks.

How does RPS6KA1 phosphorylation connect to broader questions in cellular regulation and disease pathways?

The study of RPS6KA1 phosphorylation has broader implications:

• Integration of multiple signaling pathways:

  • RPS6KA1 represents a convergence point between MAPK/ERK signaling and translation regulation

  • Its phosphorylation status reflects the integration of multiple upstream signals

• Metabolic regulation connections:

  • The role of RPS6KA1 in phosphorylating RPS6 links it to cellular energy sensing and metabolic control

  • The dramatic difference in pRPS6 between cell types with different metabolic demands (like rod and cone photoreceptors) highlights this connection

• Therapeutic targeting relevance:

  • Understanding the specific roles of RPS6KA1 phosphorylation could inform more precise therapeutic strategies

  • Distinguishing RPS6KA1-mediated effects from RPS6KB1-mediated effects could reduce off-target effects in pathway-targeted therapies

• Developmental biology insights:

  • The observed differences in pathway activation across developmental lineages suggest stage-specific roles for RPS6KA1 signaling

  • This has implications for understanding developmental disorders and directed differentiation protocols

• Cancer biology applications:

  • Aberrant RPS6KA1 phosphorylation is implicated in several cancers

  • Phospho-specific antibodies provide tools to assess pathway activation in patient samples

These connections demonstrate how detailed molecular understanding of RPS6KA1 phosphorylation contributes to addressing fundamental questions in biology and medicine.