RPS6KA4 Antibody

What is RPS6KA4 and what cellular functions does it serve in research contexts?

RPS6KA4 (ribosomal protein S6 kinase A4), also known as MSK2 or RSK-B, is a serine/threonine protein kinase that plays critical roles in signal transduction pathways. This approximately 86 kDa protein functions in the phosphorylation of multiple substrates, including transcription factors CREB1 and ATF1, in response to mitogenic or stress stimuli such as UV-C irradiation and epidermal growth factor (EGF) . RPS6KA4 contributes to gene activation through histone phosphorylation, particularly at Ser-10 and potentially Ser-28 of histone H3, resulting in transcriptional activation of immediate early genes including proto-oncogenes c-fos and c-jun . It also plays essential roles in the control of RELA transcriptional activity in response to TNF and mediates mitogen- and stress-induced phosphorylation of high mobility group protein 1 . In inflammatory contexts, RPS6KA4 acts downstream of TLR4 in lipopolysaccharide-stimulated primary macrophages . Understanding these functions is fundamental when designing experiments targeting RPS6KA4-mediated cellular processes.

What types of RPS6KA4 antibodies are commercially available for research applications?

Most commercially available RPS6KA4 antibodies are polyclonal antibodies raised in rabbits, though monoclonal options also exist. The following table summarizes key characteristics of available RPS6KA4 antibodies based on the search results:

Both antibody formats undergo purification processes, typically using protein G column chromatography or antigen affinity purification . When selecting an antibody, researchers should consider the specific experimental application, species compatibility, and validation data provided by manufacturers to ensure optimal performance.

How should RPS6KA4 antibodies be optimally stored and handled to maintain reactivity?

Proper storage of RPS6KA4 antibodies is critical for maintaining their reactivity and specificity. Most manufacturers recommend storing these antibodies at -20°C, where they remain stable for approximately one year after shipment . The antibodies are typically provided in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles . For many commercial preparations, aliquoting is unnecessary for -20°C storage, which simplifies handling protocols .

When working with these antibodies, researchers should:

• Minimize freeze-thaw cycles to prevent degradation

• Briefly centrifuge vials before opening to collect all liquid at the bottom

• Allow antibodies to reach room temperature before use in experiments

• Handle with appropriate protective equipment, especially for preparations containing sodium azide

• For smaller volume preparations (20μl), be aware that some may contain 0.1% BSA as a stabilizer

Following these handling protocols ensures maximum antibody performance and experimental reproducibility when targeting RPS6KA4.

What are the optimal technical parameters for Western blot detection of RPS6KA4?

When performing Western blot for RPS6KA4 detection, several technical considerations are critical for successful results:

• Sample Preparation: When lysing cells/tissues, include phosphatase inhibitors to preserve phosphorylation states of RPS6KA4, which is important for studying its activation state.

• Protein Loading: Load 20-50 μg of total protein per lane, as RPS6KA4 has a calculated molecular weight of 86 kDa .

• Gel Percentage: Use 8-10% SDS-PAGE gels for optimal separation of proteins in the 80-90 kDa range.

• Transfer Conditions: For proteins of this size, use wet transfer methods with methanol-containing buffer to ensure complete transfer.

• Blocking Conditions: Use 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature.

• Primary Antibody Incubation: Dilute antibody according to manufacturer's recommendations (typically 1:500 to 1:2000) in blocking buffer and incubate overnight at 4°C.

• Detection Methods: Both chemiluminescence and fluorescence-based detection systems work well, with the former being more common due to accessibility.

• Controls: Include positive controls (cell lines known to express RPS6KA4) and consider using siRNA knockdown samples as negative controls to validate specificity.

These parameters should be optimized for each specific RPS6KA4 antibody and experimental system to achieve optimal signal-to-noise ratios and reproducible results.

How can researchers distinguish between RPS6KA4 (MSK2) and other closely related RSK family members?

Distinguishing between RPS6KA4 and other RSK family members (RSK1/RPS6KA1, RSK2/RPS6KA3, RSK3/RPS6KA2, RSK4/RPS6KA6, and MSK1/RPS6KA5) requires careful experimental design due to their structural similarities. Research from ESCC studies demonstrates that these kinases have distinct expression patterns and functional roles .

Methodological approaches for differentiation:

• Antibody Selection: Choose antibodies raised against unique epitopes specific to RPS6KA4. Verify the immunogen information - antibodies generated against the C-terminal region often provide better specificity.

• RNA Expression Analysis: Utilize qPCR with gene-specific primers to quantify mRNA levels of different RSK family members. In ESCC studies, researchers demonstrated that while RPS6KA6 (RSK4) showed significant upregulation, RPS6KA1, RPS6KA3, and RPS6KA4 expression levels remained relatively unchanged .

• siRNA/shRNA Validation: Employ targeted knockdown of RPS6KA4 to confirm antibody specificity. If signals persist after RPS6KA4 knockdown, cross-reactivity with other family members may be occurring.

• Phospho-specific Detection: Different RSK family members exhibit distinct phosphorylation patterns. Using phospho-specific antibodies can help identify activation of specific family members.

• Subcellular Localization: Immunofluorescence studies can reveal differential localization patterns, as RPS6KA4/MSK2 shows distinct nuclear localization upon activation compared to some other family members.

The table below outlines key distinguishing features among RSK family members based on current research:

By employing these approaches, researchers can effectively distinguish RPS6KA4 from other RSK family members in their experimental systems .

What methodological approaches are optimal for studying RPS6KA4's role in histone modification?

RPS6KA4/MSK2 plays a critical role in histone modification, particularly through phosphorylation of histone H3 at Ser-10 and potentially Ser-28, which facilitates transcriptional activation of immediate early genes . To study this function effectively, researchers should consider the following methodological approaches:

• Chromatin Immunoprecipitation (ChIP): Use RPS6KA4 antibodies in conjunction with histone H3 phospho-Ser10 antibodies to identify genomic regions where RPS6KA4 mediates histone phosphorylation. This allows for mapping of RPS6KA4 activity across the genome.

• Dual Immunofluorescence: Co-stain cells with antibodies against RPS6KA4 and phospho-H3-Ser10 to visualize co-localization in response to stimuli such as EGF or stress conditions.

• Kinase Assays: Perform in vitro kinase assays using immunoprecipitated RPS6KA4 and recombinant histone H3 substrates to directly assess phosphorylation activity and specificity.

• Pharmacological Inhibition: Use specific inhibitors of upstream kinases (such as p38 MAPK and ERK1/2) to dissect the signaling pathways leading to RPS6KA4 activation and subsequent histone phosphorylation.

• Gene Expression Analysis: Combine RPS6KA4 manipulation (overexpression or knockdown) with RNA-seq to identify genes whose expression is regulated by RPS6KA4-mediated histone phosphorylation.

• Mass Spectrometry: Use quantitative phospho-proteomics to identify novel histone and non-histone substrates of RPS6KA4, beyond the established targets like histone H3.

These approaches, used in combination, provide robust data on RPS6KA4's role in chromatin modification and gene expression regulation, which is essential for understanding its function in various cellular contexts .

How can researchers investigate RPS6KA4's involvement in cancer radioresistance mechanisms?

Evidence from esophageal squamous cell carcinoma (ESCC) research suggests that RSK family kinases, particularly RSK4 (RPS6KA6), are involved in cancer radioresistance . While this directly focuses on RSK4 rather than RPS6KA4/MSK2, the methodological approaches are applicable for investigating RPS6KA4's potential roles in radioresistance:

• Expression Analysis in Radioresistant Models: Compare RPS6KA4 expression levels between radioresistant and radiosensitive cancer cell lines. Similar to how researchers found elevated RSK4 in ESCC after radiotherapy, researchers could analyze RPS6KA4 expression patterns before and after radiation treatment .

• Clonogenic Survival Assays: Manipulate RPS6KA4 expression (overexpression or knockdown) in cancer cells and assess colony formation ability following radiation exposure to directly measure effects on radioresistance.

• DNA Damage Response Assessment: Evaluate the impact of RPS6KA4 on DNA damage checkpoint responses by monitoring markers such as γ-H2AX levels after irradiation, similar to approaches used to study RSK4 .

• Cancer Stem Cell Property Analysis: Investigate whether RPS6KA4 influences cancer stem cell (CSC) properties through sphere formation assays and expression analysis of CSC markers like CD90, CD271, and ALDH1, drawing parallels to RSK4 studies .

• Patient Sample Analysis: Analyze patient samples before and after radiotherapy to assess RPS6KA4 expression changes and correlate with treatment outcomes and survival data.

• Signaling Pathway Investigation: Examine RPS6KA4's effects on specific signaling pathways implicated in radioresistance, such as DNA repair mechanisms or anti-apoptotic signaling.

The table below outlines key parameters for designing radioresistance studies based on RSK4 research approaches:

By adapting these approaches from RSK4 studies, researchers can systematically investigate whether RPS6KA4 plays similar or distinct roles in cancer radioresistance mechanisms .

What are common challenges in RPS6KA4 antibody applications and how can they be resolved?

When working with RPS6KA4 antibodies, researchers may encounter several technical challenges that can impact experimental outcomes. Based on the technical information available about these antibodies, here are common issues and their potential solutions:

• Non-specific Binding in Western Blots:

  • Problem: Multiple bands appearing around the expected 86 kDa molecular weight .

  • Solution: Optimize blocking conditions (try 5% BSA instead of milk for phospho-specific detection), increase washing stringency, and dilute primary antibody further. Consider using gradient gels for better separation of proteins in the 80-90 kDa range.

• Weak Signal in Immunohistochemistry:

  • Problem: Low or undetectable staining in tissues known to express RPS6KA4.

  • Solution: Optimize antigen retrieval methods (try different pH buffers and heating times), increase antibody concentration, and extend incubation times. Consider signal amplification systems for low-abundance targets.

• Background in Immunofluorescence:

  • Problem: High non-specific fluorescence reducing signal-to-noise ratio.

  • Solution: Use more stringent blocking with normal serum from the secondary antibody species, include 0.1-0.3% Triton X-100 for better antibody penetration, and optimize secondary antibody concentration.

• Cross-reactivity with Related Proteins:

  • Problem: Difficulty distinguishing between RPS6KA4 and other RSK family members.

  • Solution: Validate specificity using knockout/knockdown controls, perform peptide competition assays, or switch to antibodies raised against unique epitopes specific to RPS6KA4.

• Variable Results Across Sample Types:

  • Problem: Inconsistent detection across different species or tissue types.

  • Solution: Confirm antibody species reactivity (human, mouse, rat) , adjust protein extraction protocols for different tissue types, and optimize fixation conditions for each sample type.

By systematically addressing these technical challenges, researchers can improve the reliability and reproducibility of experiments using RPS6KA4 antibodies.

How should researchers validate the specificity of RPS6KA4 antibodies?

Validating antibody specificity is crucial for generating reliable and reproducible data in RPS6KA4 research. Based on best practices in antibody validation, researchers should implement the following approaches:

• Genetic Controls:

  • Use RPS6KA4 knockout cell lines created through CRISPR-Cas9 editing as negative controls.

  • Alternatively, employ siRNA/shRNA knockdown to reduce RPS6KA4 expression and confirm corresponding reduction in antibody signal.

• Overexpression Systems:

  • Express tagged RPS6KA4 (e.g., with FLAG or HA) in appropriate cell systems and confirm co-localization with the RPS6KA4 antibody signal.

  • Use non-transfected cells as controls to establish baseline detection levels.

• Peptide Competition Assays:

  • Pre-incubate the RPS6KA4 antibody with excess immunizing peptide (if available) before application to samples.

  • Valid antibodies will show significantly reduced or abolished signal when the specific epitope is blocked.

• Multi-antibody Validation:

  • Compare results using different antibodies that recognize distinct epitopes within RPS6KA4.

  • Consistent patterns across different antibodies increase confidence in specificity.

• Expression Pattern Verification:

  • Compare antibody detection patterns with mRNA expression data from public databases or RT-qPCR experiments.

  • Discrepancies between protein and mRNA patterns may indicate antibody specificity issues.

• Mass Spectrometry Validation:

  • Perform immunoprecipitation using the RPS6KA4 antibody followed by mass spectrometry analysis.

  • This confirms whether the antibody is pulling down RPS6KA4 and identifies any cross-reactive proteins.

• Phosphorylation-state Specificity:

  • For phospho-specific antibodies, treat samples with phosphatases to confirm the signal is dependent on phosphorylation status.

  • Use activators of the MAPK pathway to increase RPS6KA4 phosphorylation as positive controls.

These validation steps should be documented and included in publications to enhance transparency and reproducibility in RPS6KA4 research.

How can RPS6KA4 antibodies be utilized to investigate inflammatory signaling pathways?

RPS6KA4/MSK2 plays important roles in inflammatory signaling, particularly downstream of TLR4 in lipopolysaccharide-stimulated macrophages . Researchers can leverage RPS6KA4 antibodies to investigate these inflammatory pathways through several methodological approaches:

• Phospho-specific Detection of Inflammatory Transcription Factors:

  • Use antibodies against both RPS6KA4 and its phosphorylated targets (CREB1, ATF1, and RELA) to map activation kinetics following inflammatory stimuli.

  • Combine with inhibitors of upstream kinases (p38 MAPK, ERK1/2) to delineate specific signaling cascades.

• Chromatin Immunoprecipitation Sequencing (ChIP-seq):

  • Employ RPS6KA4 antibodies in ChIP-seq experiments to identify genomic regions where RPS6KA4 binds following inflammatory activation.

  • Correlate with histone H3 phosphorylation patterns to understand epigenetic regulation of inflammatory gene expression.

• Co-immunoprecipitation Studies:

  • Use RPS6KA4 antibodies to immunoprecipitate the protein complex and identify interaction partners specific to inflammatory contexts.

  • This can reveal novel components of inflammatory signaling complexes that modulate gene expression.

• Tissue-specific Expression Profiling:

  • Apply immunohistochemistry using RPS6KA4 antibodies to map expression patterns in inflamed versus healthy tissues.

  • Correlate with markers of inflammation to understand the relationship between RPS6KA4 expression and inflammatory states.

• Single-cell Analysis:

  • Combine RPS6KA4 antibodies with other inflammatory markers in multiparameter flow cytometry or mass cytometry to examine cell-specific responses in heterogeneous populations.

  • This can reveal differential RPS6KA4 activation across immune cell subsets during inflammatory responses.

• Live-cell Imaging:

  • Use fluorescently tagged antibody fragments to monitor RPS6KA4 dynamics in live cells responding to inflammatory stimuli.

  • This approach can reveal temporal and spatial regulation of RPS6KA4 activity.

These methodologies enable detailed investigation of RPS6KA4's functions in inflammatory signaling networks, potentially identifying novel therapeutic targets for inflammatory diseases .

What is the current understanding of RPS6KA4's role in cellular stress responses?

RPS6KA4/MSK2 serves as a key mediator in cellular stress response pathways, integrating signals from various stressors to orchestrate appropriate transcriptional responses. Current research indicates several important aspects of RPS6KA4's role in stress responses:

• Activation Mechanisms: RPS6KA4 is activated in response to multiple stress stimuli, including UV-C irradiation and anisomycin . This activation occurs through phosphorylation by upstream kinases in the MAPK pathway, primarily p38 MAPK and ERK1/2, positioning RPS6KA4 as an integration point for diverse stress signals.

• Transcriptional Regulation: Upon activation, RPS6KA4 phosphorylates transcription factors including CREB1 and ATF1 , which then regulate the expression of stress-responsive genes. This transcriptional programming is critical for appropriate cellular adaptation to stress conditions.

• Chromatin Modification: RPS6KA4 mediates stress-induced phosphorylation of histone H3 at Ser-10 , which facilitates chromatin remodeling and accessibility of stress-responsive gene promoters. This epigenetic regulation represents a key mechanism by which RPS6KA4 influences the transcriptional landscape during stress responses.

• Immediate Early Gene Induction: Through its actions on chromatin and transcription factors, RPS6KA4 contributes to the rapid induction of immediate early genes such as c-fos and c-jun , which are pivotal for initiating broader stress response programs.

• Inflammatory Stress Response: In contexts of inflammatory stress, RPS6KA4 acts downstream of TLR4 signaling to regulate inflammatory gene expression , suggesting a role in modulating immune responses to pathogenic and environmental stressors.

To effectively study these aspects of RPS6KA4 function, researchers should design experiments that:

• Compare RPS6KA4 activation across different stress modalities (oxidative, genotoxic, inflammatory)

• Examine temporal dynamics of RPS6KA4 activation and downstream effects

• Investigate cell type-specific differences in RPS6KA4-mediated stress responses

• Assess the consequences of RPS6KA4 inhibition or depletion on cellular survival and adaptation to stress

Understanding RPS6KA4's multifaceted roles in stress responses has implications for numerous pathological conditions, including cancer, inflammatory disorders, and neurodegenerative diseases where stress response pathways are dysregulated.