Phospho-RPS6KA1 (Ser352) Antibody

What is RPS6KA1 and why is phosphorylation at Ser352 significant?

RPS6KA1 (RSK1) is a growth-factor regulated serine/threonine kinase involved in the MAPK cascade. It controls cellular proliferation and differentiation through phosphorylation of transcription factors. RPS6KA1 contains two nonidentical kinase catalytic domains and phosphorylates various substrates, including members of the mitogen-activated kinase (MAPK) signaling pathway .

Phosphorylation at Ser352 is part of the complex activation mechanism of RPS6KA1. While specific phosphorylation sites including Ser380, Thr359, and Ser363 have been well-characterized as important for kinase activation , Ser352 represents another regulatory site involved in controlling the kinase activity of RPS6KA1.

What applications are Phospho-RPS6KA1 (Ser352) antibodies commonly used for?

Phospho-specific RPS6KA1 antibodies are typically used in the following applications:

Similar to other phospho-specific antibodies like the Phospho-RPS6KA1 (Ser380), Phospho-RPS6KA1 (Ser352) antibodies should be validated for specificity and optimal conditions in your experimental system .

How does RPS6KA1 function in cellular signaling pathways?

RPS6KA1 functions as a key downstream effector in the MAPK cascade. When activated:

• It is directly phosphorylated by ERK1/2 following growth factor stimulation

• This leads to autophosphorylation and full activation of RPS6KA1

• Activated RPS6KA1 then translocates to the nucleus

• Within the nucleus, it phosphorylates various transcription factors including CREB1, ETV1/ER81, and NR4A1/NUR77

This signaling cascade is critical for mediating cellular processes including proliferation, survival, and differentiation. RPS6KA1 essentially acts as a bridge between cytoplasmic signaling events and nuclear transcriptional regulation .

What is the optimal protocol for detecting phosphorylated RPS6KA1 (Ser352) via Western blotting?

For optimal detection of phosphorylated RPS6KA1 at Ser352, follow this methodological approach:

• Sample preparation:

  • Harvest cells at log phase (1×10^5 to 1×10^6 cells/ml)

  • Lyse cells in RIPA buffer supplemented with both protease AND phosphatase inhibitors

  • Determine protein concentration using a BCA protein assay kit

• SDS-PAGE and transfer:

  • Load equal amounts of protein (25-50 μg)

  • Use a 10% polyacrylamide gel for optimal separation

  • Transfer to nitrocellulose membrane at 100V for 60-90 minutes

• Antibody incubation:

  • Block membrane with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with Phospho-RPS6KA1 (Ser352) antibody at 1:1000 dilution overnight at 4°C

  • Wash 3× with TBST

  • Incubate with HRP-conjugated secondary antibody (1:4000) for 1 hour

• Validation controls:

  • Include λ-phosphatase treated lysate as a negative control

  • Include lysates from cells treated with activators of the MAPK pathway (e.g., PMA, EGF) as a positive control

  • Expected molecular weight: 83-90 kDa

How can I validate the specificity of a phospho-RPS6KA1 (Ser352) antibody?

Validating phospho-specific antibodies requires several complementary approaches:

• Phosphatase treatment:

  • Treat half your sample with λ-phosphatase to remove phosphorylation

  • The signal should disappear in treated samples while total RPS6KA1 remains detectable

• Phospho-blocking peptide competition:

  • Pre-incubate antibody with the phosphorylated peptide immunogen

  • The specific signal should be significantly reduced or eliminated

• Kinase activation/inhibition:

  • Treat cells with activators (EGF, PMA) or inhibitors (U0126, BI-D1870) of the MAPK/RSK pathway

  • Confirm expected changes in phosphorylation levels

• Site-directed mutagenesis:

  • Use cells expressing RPS6KA1 with Ser352 mutated to alanine (S352A)

  • The antibody should not detect the mutant protein

What cell stimulation conditions are optimal for studying RPS6KA1 phosphorylation?

For effective induction of RPS6KA1 phosphorylation:

For maximum detection of phosphorylated RPS6KA1:

• Use serum-starved cells (0.1-0.5% serum for 16-24 hours) before stimulation

• Add phosphatase inhibitors (e.g., calyculin A) before cell lysis

• Maintain samples on ice during processing to minimize dephosphorylation

How can phospho-RPS6KA1 (Ser352) antibodies be used in cancer research?

Phospho-RPS6KA1 antibodies have significant applications in cancer research, especially regarding therapy resistance:

• Biomarker development:

  • RPS6KA1 activation correlates with resistance to targeted therapies in multiple cancer types

  • Phospho-RPS6KA1 levels can be monitored as predictive biomarkers

• Drug resistance mechanisms:

  • RPS6KA1 has been identified as a mediator of resistance to venetoclax/azacitidine treatment in acute myeloid leukemia (AML)

  • Researchers can use phospho-RPS6KA1 antibodies to monitor activation status following treatment

• Combination therapy studies:

  • Addition of RPS6KA1 inhibitors (like BI-D1870) to standard therapies can decrease proliferation and increase sensitivity to treatment

  • Phospho-RPS6KA1 antibodies allow monitoring of inhibitor efficacy in both research and clinical settings

• Subpopulation analysis:

  • Using flow cytometry with phospho-RPS6KA1 antibodies can help identify therapy-resistant cell subpopulations

  • Particularly effective for studying monocytic blast subclones in AML, which may be sources of relapse

What are the considerations for multiplexing phospho-RPS6KA1 detection with other signaling markers?

When designing multiplexed studies involving phospho-RPS6KA1:

• Antibody compatibility:

  • Ensure primary antibodies are from different host species (e.g., rabbit anti-phospho-RPS6KA1 with mouse anti-ERK)

  • Or use directly conjugated primary antibodies with distinct fluorophores

• Signal separation:

  • For immunofluorescence or flow cytometry, choose fluorophores with minimal spectral overlap

  • For chemiluminescent Western blots, consider sequential detection with stripping or multiplex fluorescent detection

• Pathway relationships:

  • Pair phospho-RPS6KA1 with upstream regulators (phospho-ERK1/2) and downstream targets (phospho-S6, phospho-CREB)

  • This approach provides a comprehensive view of pathway activation status

• Temporal dynamics:

  • Different phosphorylation events occur with distinct kinetics

  • Design time-course experiments that capture the optimal window for each phosphorylation site

How do I troubleshoot weak or non-specific signals when using phospho-RPS6KA1 (Ser352) antibodies?

When troubleshooting phospho-specific antibody issues:

• Weak signal:

  • Ensure sufficient pathway activation (use positive controls like calyculin A-treated cells)

  • Increase antibody concentration or incubation time

  • Use enhanced sensitivity detection methods (e.g., enhanced chemiluminescence)

  • Verify sample handling preserves phosphorylation (phosphatase inhibitors, cold processing)

• High background/non-specific bands:

  • Increase blocking time and concentration (5% BSA in TBST)

  • Reduce primary antibody concentration

  • Include phospho-blocking peptides as competitors for non-specific binding

  • Try alternative secondary antibodies

  • Increase washing duration and number of washes

• Multiple bands:

  • Verify molecular weight (83-90 kDa for full-length RPS6KA1)

  • Test specificity with phosphatase treatment

  • Consider the presence of isoforms or degradation products

  • Evaluate possible cross-reactivity with other RSK family members

How can proximity ligation assays be used with phospho-RPS6KA1 antibodies to study protein interactions?

Proximity Ligation Assay (PLA) with phospho-RPS6KA1 antibodies offers powerful insights:

• Methodology:

  • Requires two antibodies: one against phospho-RPS6KA1 and one against a potential interaction partner

  • Each red dot in the assay represents a single phosphorylated protein interaction

  • Optimal dilutions: rabbit polyclonal antibody (1:1200) and mouse monoclonal antibody (1:50)

• Applications:

  • Detect interactions between phosphorylated RPS6KA1 and downstream substrates

  • Study subcellular localization of active RPS6KA1 complexes

  • Quantify activation states of individual RPS6KA1 molecules

• Data analysis:

  • Images can be analyzed using BlobFinder software from Uppsala University

  • Results provide quantitative data on interaction frequency

PLA is particularly valuable because it provides spatial information about protein interactions at the single-molecule level, something traditional co-immunoprecipitation cannot offer.

What are the latest approaches for studying the temporal dynamics of RPS6KA1 phosphorylation?

Advanced techniques for temporal phosphorylation analysis include:

• Real-time kinetic measurements:

  • Microarray-based kinetic colorimetric detection allows for multiplex protein phosphorylation analysis

  • This approach enables quantitative tracking of phosphorylation events over time

• Phosphoproteomics integration:

  • Mass spectrometry-based phosphoproteomics can identify multiple phosphorylation sites simultaneously

  • Sample preparation includes cell lysis, protein reduction with DTT, alkylation with CAA, and digestion with trypsin

  • Analysis on a Tri-Hybrid Orbitrap Fusion mass spectrometer with MaxQuant processing

• FRET-based biosensors:

  • Genetically encoded biosensors can monitor RPS6KA1 activity in living cells

  • These provide real-time visualization of phosphorylation dynamics

• Single-cell phospho-flow cytometry:

  • Enables analysis of phosphorylation heterogeneity within cell populations

  • Particularly valuable for studying cancer cell subpopulations with differential drug responses

How does phosphorylation at Ser352 relate to other phosphorylation sites on RPS6KA1?

Understanding the relationship between different phosphorylation sites is crucial:

The activation of RPS6KA1 involves a sequential phosphorylation cascade:

• ERK1/2 phosphorylates Thr359/Ser363 in the linker region and Thr573 in the C-terminal kinase domain (CTKD)

• This activates the CTKD, which then autophosphorylates Ser380 and potentially Ser352

• Phosphorylated Ser380 creates a docking site for PDK1

• PDK1 then phosphorylates Ser221 in the N-terminal kinase domain (NTKD)

• The fully activated NTKD can then phosphorylate substrates

How can phospho-RPS6KA1 antibodies be used to study acute myeloid leukemia (AML) resistance mechanisms?

Phospho-specific RPS6KA1 antibodies have been instrumental in elucidating resistance mechanisms in AML:

• Experimental approaches:

  • Genome-wide CRISPR/Cas9 screens identified RPS6KA1 as a mediator of resistance to venetoclax/azacitidine treatment

  • Phospho-RPS6KA1 antibodies enable monitoring of activation status in response to therapy

  • Western blotting with these antibodies can confirm efficacy of RPS6KA1 inhibitors like BI-D1870

• Clinical relevance:

  • RPS6KA1 inhibition with BI-D1870 restored sensitivity in venetoclax/azacitidine-resistant AML cells

  • Measurement of phospho-RPS6KA1 levels can potentially identify patients likely to develop resistance

  • RPS6KA1 inhibition efficiently targeted monocytic blast subclones, a potential source of relapse

• Methodology:

  • Cell viability assays with inhibitor combinations can be read out with CellTiter 96 AQueous One Solution

  • Flow cytometric analysis using phospho-RPS6KA1 antibodies can identify resistant subpopulations

  • Colony formation assays provide functional validation of inhibitor effectiveness

This research demonstrates how phospho-specific antibodies can connect molecular mechanisms to potential therapeutic interventions.

What are best practices for optimizing immunohistochemistry with phospho-RPS6KA1 antibodies?

For successful immunohistochemistry with phospho-RPS6KA1 antibodies:

• Fixation and preservation:

  • Use phosphatase inhibitors during tissue collection and processing

  • Fresh frozen samples often provide better phospho-epitope preservation than FFPE

  • If using FFPE, limit fixation time to preserve phospho-epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Include phosphatase inhibitors in all buffers

  • Optimize retrieval time (typically 15-20 minutes)

• Signal amplification:

  • Consider tyramide signal amplification for low abundance phospho-epitopes

  • Use polymer-based detection systems for improved sensitivity

  • Optimize antibody concentration (typically 1:100-1:500)

• Controls:

  • Include phosphatase-treated sections as negative controls

  • Use tissues from pathway-activated models as positive controls

  • Always run both phospho-specific and total protein antibodies on adjacent sections

How can I design multiplexed mass cytometry panels to include phospho-RPS6KA1?

For effective mass cytometry (CyTOF) panel design:

• Metal conjugation:

  • Select rare earth metals for phospho-RPS6KA1 antibodies (e.g., 151Eu, 153Eu)

  • Perform validation of metal-conjugated antibodies against unconjugated versions

  • Titrate antibodies to determine optimal concentration

• Panel design considerations:

  • Include upstream (phospho-ERK1/2) and downstream (phospho-S6) markers

  • Add markers for cell lineage identification and functional status

  • Include barcoding channels for sample multiplexing

• Sample preparation:

  • Fix cells immediately after stimulation (2% paraformaldehyde)

  • Permeabilize with ice-cold methanol to preserve phospho-epitopes

  • Include both unstimulated and strongly stimulated controls

• Data analysis:

  • Use visualization tools like viSNE or UMAP for high-dimensional data

  • Apply phospho-flow gating strategies to identify pathway-activated subpopulations

  • Consider algorithms like DREMI to quantify signaling relationships

What are the considerations for using phospho-RPS6KA1 antibodies in patient-derived samples?

Working with patient samples requires special considerations:

• Pre-analytical variables:

  • Standardize time from collection to fixation (<30 minutes ideal)

  • Document ischemia time as this affects phosphorylation status

  • Consider the impact of prior treatments on phosphorylation levels

• Technical optimization:

  • Validate antibodies specifically on patient-derived material

  • Determine optimal fixation and permeabilization conditions for specific sample types

  • Use matched normal tissues as controls when possible

• Clinical correlation:

  • Correlate phospho-RPS6KA1 levels with treatment response

  • Consider analysis in the context of other molecular markers

  • Evaluate potential as a predictive biomarker for RPS6KA1 inhibitor therapy

• Ethical and regulatory considerations:

  • Ensure appropriate informed consent and IRB approval

  • Consider preanalytical standardization for biomarker development

  • Document chain of custody for clinical samples