Phospho-SOX9 (Ser181) Antibody

What specific epitope does the Phospho-SOX9 (Ser181) antibody recognize?

The antibody specifically recognizes SOX9 protein only when phosphorylated at Serine 181. It is typically generated using synthetic phosphopeptides derived from human SOX9 around the phosphorylation site of Serine 181, usually within the amino acid region of 147-196 . This specificity ensures that the antibody distinguishes between the phosphorylated and non-phosphorylated forms of SOX9, making it valuable for studying the activation state of this transcription factor .

What is the molecular significance of SOX9 phosphorylation at Ser181?

Phosphorylation of SOX9 at Ser181 enhances its transcriptional activity and is a key regulatory event in multiple biological processes. This modification is particularly important because it occurs near the HMG box domain, which mediates DNA binding . Research has shown that this phosphorylation event increases SOX9's ability to activate target genes involved in chondrogenesis, including cartilage matrix protein-coding genes such as COL2A1, COL4A2, COL9A1, COL11A2, and ACAN . Additionally, Ser181 phosphorylation promotes nuclear accumulation of SOX9, thereby enhancing its function as a transcription factor .

Which kinases are responsible for phosphorylating SOX9 at Ser181?

Several kinases have been identified that can phosphorylate SOX9 at Ser181:

These diverse kinases highlight the central role of SOX9 Ser181 phosphorylation as an integration point for multiple signaling pathways regulating development and cellular differentiation .

How does phosphorylation at Ser181 regulate SOX9's interaction with other proteins and DNA?

Phosphorylation of SOX9 at Ser181 occurs near its HMG box domain, which is responsible for DNA binding . This modification enhances SOX9's ability to bind to the 5'-ACAAAG-3' DNA motif present in enhancers and super-enhancers of target genes . Research suggests that this phosphorylation may modulate DNA interactions either by changing the conformation of the HMG box or by recruiting additional cofactors .

In terms of protein interactions, phosphorylated SOX9 shows enhanced binding to transcriptional co-activators. For example, studies in glomerular mesangial cells revealed that ERK1/2 not only phosphorylates SOX9 at Ser181 but also physically interacts with phosphorylated SOX9 in the nucleus, forming a complex that promotes Cyclin D1 gene transcription . This demonstrates how Ser181 phosphorylation can serve as a platform for assembling transcriptional complexes that drive specific gene expression programs.

What are the established protocols for validating Phospho-SOX9 (Ser181) antibody specificity in experimental systems?

Validating the specificity of Phospho-SOX9 (Ser181) antibody is crucial for obtaining reliable results. Several approaches have been documented:

• Peptide Competition Assay: Treating the antibody with the immunogenic phosphopeptide before application to samples should block specific binding. This approach has been used to confirm specificity, as shown in Figure 2 of source , where a single protein band was detected that could be blocked by the synthesized immunogen peptide .

• Phospho-specific ELISA: Comparing antibody binding to phosphorylated versus non-phosphorylated peptides demonstrates specificity for the phosphorylated form. For example, Figure 3 in source shows that the Anti-SOX9 (Phospho-Ser181) Antibody is highly specific for the phospho-peptide with minimal binding to the non-phospho counterpart .

• Phosphatase Treatment: Treating samples with lambda phosphatase before immunoblotting should eliminate the signal if the antibody is truly phospho-specific.

• Kinase Activation/Inhibition: Stimulating samples with known activators of SOX9 phosphorylation (e.g., PMA treatment of HeLa cells ) or treating with specific kinase inhibitors can confirm the antibody's ability to detect dynamic changes in phosphorylation status.

How do experimental conditions affect the detection of Phospho-SOX9 (Ser181)?

Several experimental factors can significantly impact the detection of Phospho-SOX9 (Ser181):

• Sample Preparation: Rapid sample processing is essential as phosphorylation states can change quickly. Samples should be collected in buffer containing phosphatase inhibitors to prevent dephosphorylation during processing .

• Fixation Methods: For immunohistochemistry, the choice of fixative is critical. Formaldehyde (4% for adherent cells, 8% for suspension cells) has been recommended for optimal preservation of phospho-epitopes .

• Blocking Conditions: Using 5% BSA rather than milk is recommended for Western blotting, as milk contains phosphoproteins that may increase background .

• Detection Systems: For low abundance phosphoproteins, enhanced chemiluminescence or fluorescence-based detection systems may provide better sensitivity than colorimetric methods.

• Cell Density and Treatment Timing: The phosphorylation of SOX9 at Ser181 is dynamic and can be influenced by cell confluency, serum conditions, and treatment duration. For example, in HeLa cells, PMA treatment (100nM for 30 minutes) has been shown to increase Ser181 phosphorylation .

What is the relationship between SOX9 Ser181 phosphorylation and disease pathology?

Abnormal SOX9 Ser181 phosphorylation has been implicated in several pathological conditions:

• Mesangioproliferative Glomerulonephritis (MsPGN): Studies have shown increased levels of phosphorylated SOX9 (Ser181) in renal tissues of MsPGN patients, with a positive correlation between p-ERK1/2, p-SOX9, and Cyclin D1 expression (r² = 0.5425, p < 0.01) . This suggests that aberrant SOX9 phosphorylation contributes to abnormal cell proliferation in this renal disease.

• Cartilage Disorders: Since SOX9 is a master regulator of chondrogenesis, dysregulation of its phosphorylation at Ser181 may contribute to skeletal disorders. ROCK-dependent phosphorylation of SOX9 at Ser181 increases in response to TGF-β treatment and mechanical compression, suggesting a role in mechanotransduction and potential involvement in osteoarthritis pathogenesis .

• Cancer: Increased nuclear localization of phosphorylated SOX9 has been observed in various cancers, including colon carcinoma . The phosphorylation status of SOX9 may influence cancer cell proliferation and invasion through regulation of target genes such as Cyclin D1.

What are the optimal protocols for Western blot analysis using Phospho-SOX9 (Ser181) antibody?

For optimal Western blot results with Phospho-SOX9 (Ser181) antibody:

• Sample Preparation:

  • Lyse cells in RIPA buffer containing phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Maintain samples at 4°C during processing

  • Standardize protein quantification, typically loading 20-50 μg per lane

• Gel Electrophoresis and Transfer:

  • Use 10% SDS-PAGE gels for optimal resolution around 56 kDa (the molecular weight of SOX9)

  • Transfer to PVDF membrane (preferred over nitrocellulose for phosphoproteins)

• Blocking and Antibody Incubation:

  • Block with 5% BSA (not milk) in TBST for 1 hour at room temperature

  • Dilute primary antibody 1:500-1:2000 in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle agitation

  • Perform thorough washes (3-5 times, 5 minutes each) with TBST

• Detection:

  • Use HRP-conjugated secondary antibodies (1:5000-1:10000)

  • Enhanced chemiluminescence detection is recommended for sensitivity

• Controls:

  • Include positive controls: PMA-treated HeLa lysates (100nM, 30min), mouse brain tissue lysates, or rat liver tissue lysates have been validated

  • Include negative controls: untreated samples or samples where phosphorylation should be absent

The antibody detects endogenous SOX9 protein at approximately 56 kDa when phosphorylated at Ser181, with >95% purity by SDS-PAGE .

How should researchers optimize immunohistochemistry protocols for Phospho-SOX9 (Ser181) detection?

For optimal immunohistochemistry results:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Paraffin embedding should follow standard protocols

  • Section thickness of 4-6 μm is recommended

• Antigen Retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Allow slides to cool slowly to room temperature

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Block non-specific binding with 5% normal goat serum

  • Dilute antibody 1:50-1:300 in blocking solution

  • Incubate overnight at 4°C in a humidified chamber

• Detection:

  • Use biotin-streptavidin or polymer-based detection systems

  • DAB (3,3'-diaminobenzidine) is commonly used as chromogen

  • Counterstain with hematoxylin for nuclear visualization

• Controls and Validation:

  • Positive control: Human colon carcinoma tissue has been validated to show nuclear staining

  • Negative control: Replace primary antibody with PBS or non-immune IgG

  • Phosphorylated SOX9 (Ser181) typically shows nuclear localization in positive cells

What are the key considerations for designing experiments to study dynamic changes in SOX9 Ser181 phosphorylation?

When designing experiments to study dynamic changes in SOX9 Ser181 phosphorylation:

• Time Course Analysis:

  • Include multiple time points (e.g., 0, 15, 30, 60, 120, 180 minutes) to capture the kinetics of phosphorylation

  • In vitro studies show that SOX9 phosphorylation at Ser181 often peaks around 3 hours after stimulation

• Stimulation Protocols:

  • Well-characterized stimuli include:

    • PMA (100nM) for PKC pathway activation

    • TGF-β for ROCK pathway activation

    • Mechanical compression for chondrocytes

    • Sublytic C5b-9 for glomerular mesangial cells

• Inhibitor Studies:

  • Use specific kinase inhibitors to block phosphorylation:

    • Y-27632 for ROCK inhibition

    • U0126 for MEK/ERK inhibition

    • H-89 for PKA inhibition

• Quantification Methods:

  • Western blot: Normalize phospho-SOX9 signal to total SOX9

  • Immunofluorescence: Measure nuclear/cytoplasmic ratio of phospho-SOX9

  • Cell-Based ELISA: Normalize to total cell number using Crystal Violet staining

• Functional Correlation:

  • Correlate changes in phosphorylation with functional outcomes:

    • Transcriptional activity using luciferase reporter assays

    • Target gene expression (COL2A1, ACAN, etc.)

    • Cell proliferation, as measured by Cyclin D1 expression or BrdU incorporation

What approaches can be used to simultaneously study multiple post-translational modifications of SOX9?

SOX9 undergoes several post-translational modifications besides Ser181 phosphorylation. To study these modifications simultaneously:

• Sequential Immunoprecipitation:

  • First IP with one modification-specific antibody

  • Elute and perform second IP with another modification-specific antibody

  • This approach can determine if modifications co-exist on the same protein molecules

• Mass Spectrometry:

  • Immunoprecipitate SOX9 and analyze by LC-MS/MS

  • This approach can identify multiple modifications simultaneously

  • Has been used to identify novel phosphorylation sites such as Ser64

• Multiplexed Western Blotting:

  • Strip and reprobe membranes with antibodies against different modifications

  • Alternatively, use fluorescently labeled secondary antibodies for simultaneous detection

• Proximity Ligation Assay (PLA):

  • Use antibodies against different modifications of SOX9

  • Can visualize co-occurrence of modifications at the single-molecule level

The table below summarizes key post-translational modifications of SOX9 that might be studied together:

How can researchers troubleshoot weak or absent signals when using Phospho-SOX9 (Ser181) antibody?

When troubleshooting weak or absent signals:

• Sample Preparation Issues:

  • Ensure phosphatase inhibitors were included during sample preparation

  • Check protein degradation by Ponceau S staining of the membrane

  • Verify total SOX9 is detectable using a non-phospho-specific antibody

• Antibody Handling:

  • Confirm antibody storage conditions (4°C short-term, -20°C long-term, avoiding freeze/thaw cycles)

  • Check antibody concentration (typically 1 mg/mL)

  • Consider using fresh aliquots if antibody has been stored for an extended period

• Technical Adjustments:

  • Increase antibody concentration or incubation time

  • Enhance signal using more sensitive detection methods

  • For IHC/IF, optimize antigen retrieval methods

• Biological Considerations:

  • Confirm SOX9 expression in your cell type/tissue

  • Verify that experimental conditions should induce Ser181 phosphorylation

  • Consider positive controls known to express phosphorylated SOX9 (e.g., PMA-treated HeLa cells)

• Methodological Alternatives:

  • Try alternative applications (e.g., if WB fails, try IHC or IP)

  • Consider enriching phosphoproteins before analysis

How should researchers interpret conflicting results between phosphorylation status and functional outcomes?

When faced with discrepancies between SOX9 Ser181 phosphorylation and expected functional outcomes:

• Consider Other Regulatory Mechanisms:

  • SOX9 activity is regulated by multiple PTMs, not just Ser181 phosphorylation

  • Check for other modifications (e.g., Ser64 phosphorylation, acetylation)

  • Assess protein-protein interactions that might modulate activity

• Examine Subcellular Localization:

  • Phosphorylation may increase, but nuclear localization could be impaired

  • Use fractionation or immunofluorescence to determine localization

• Evaluate Cellular Context:

  • Cell-type specific cofactors may be required for full activity

  • Assess expression of partner proteins (e.g., SOX5, SOX6 for chondrogenesis)

• Temporal Considerations:

  • Phosphorylation kinetics may not align with functional readouts

  • Extend time course measurements

• Dose-Response Relationships:

  • Threshold effects may exist where certain levels of phosphorylation are required

  • Quantify phosphorylation levels precisely and correlate with function

• Validation Approaches:

  • Use phosphomimetic (S181D/E) or phospho-deficient (S181A) SOX9 mutants

  • This approach can help establish causality between phosphorylation and function

What are the best practices for quantifying and normalizing Phospho-SOX9 (Ser181) levels in different experimental contexts?

For accurate quantification and normalization:

• Western Blot Analysis:

  • Always normalize phospho-SOX9 to total SOX9 from the same samples

  • Use internal loading controls (GAPDH, β-actin) for total protein normalization

  • Employ densitometry software for quantification

  • Stay within the linear range of detection

• Cell-Based ELISA:

  • Normalize to cell number using Crystal Violet staining

  • Include both phospho-SOX9 and total SOX9 antibodies in parallel wells

  • Calculate the ratio of phospho-SOX9 to total SOX9

• Immunohistochemistry/Immunofluorescence:

  • Use digital image analysis for quantification

  • Score both intensity and percentage of positive cells

  • Compare equivalent anatomical regions across samples

  • Use standardized exposure settings for all samples

• Flow Cytometry:

  • Establish clear positive and negative populations

  • Use median fluorescence intensity (MFI) for quantification

  • Include isotype controls and secondary-only controls

• Data Presentation:

  • Express results as fold change relative to control conditions

  • Include statistical analysis with appropriate tests

  • Present individual data points along with means/medians for transparency