Phospho-HSF1 (Ser307) Antibody

What is Phospho-HSF1 (Ser307) Antibody and what does it specifically detect?

Phospho-HSF1 (Ser307) Antibody is a rabbit polyclonal antibody designed to specifically detect the heat shock factor 1 (HSF1) protein only when phosphorylated at serine 307. The antibody is generated by immunizing rabbits with synthetic phosphopeptides and KLH conjugates around the phosphorylation site of serine 307 (P-Q-S(p)-P-R) derived from Human HSF1 . To ensure specificity, these antibodies undergo a two-step purification process: first through affinity-chromatography using epitope-specific phosphopeptides, and subsequently through chromatography using non-phosphopeptides to remove any non-phospho-specific antibodies . This rigorous purification process enables the antibody to selectively recognize the phosphorylated form of HSF1 at Ser307 without cross-reactivity to the non-phosphorylated form.

What is the biological function of HSF1 and the significance of its Ser307 phosphorylation?

HSF1 functions as a stress-inducible and DNA-binding transcription factor that plays a central role in the transcriptional activation of the heat shock response (HSR). This activation leads to the expression of molecular chaperones and heat shock proteins (HSPs) that protect cells from cellular damage . In unstressed cells, HSF1 is present in a HSP90-containing multichaperone complex that maintains it in a non-DNA-binding inactivated monomeric form. Upon exposure to heat and other stress stimuli, HSF1 undergoes homotrimerization and activates HSP gene transcription .

Ser307 phosphorylation represents one of several critical regulatory phosphorylation sites on HSF1. Research indicates that phosphorylation at Ser307 (along with Ser303) represses HSF1 transcriptional activity . Studies using knock-in mouse models where Ser303 and Ser307 were replaced with alanine residues demonstrated increased HSF1 activity, suggesting that these phosphorylation sites function as negative regulatory elements in HSF1-mediated transcription .

What species reactivity does the Phospho-HSF1 (Ser307) Antibody demonstrate?

The Phospho-HSF1 (Ser307) Antibody demonstrates reactivity with human, mouse, and rat samples . This cross-species reactivity makes the antibody valuable for comparative studies examining HSF1 phosphorylation status across different model organisms. The conservation of this phosphorylation site across multiple mammalian species suggests its evolutionary importance in regulating HSF1 function.

What are the validated applications for Phospho-HSF1 (Ser307) Antibody?

Based on extensive validation studies, Phospho-HSF1 (Ser307) Antibody can be reliably used for:

• Western Blot (WB): Recommended dilution ranges from 1:500 to 1:2000

• Immunofluorescence (IF): Recommended dilution ranges from 1:100 to 1:1000

• ELISA: Recommended dilution approximately 1:5000

• Immunohistochemistry (IHC): For both paraffin sections (IHC-p) and frozen sections (IHC-f)

The versatility across multiple applications makes this antibody particularly valuable for multifaceted experimental approaches that require confirmation of results through different methodologies.

How should samples be prepared for optimal detection of phosphorylated HSF1 at Ser307?

For optimal detection of phosphorylated HSF1 at Ser307, researchers should consider the following methodological guidelines:

• Sample preparation: Rapidly harvest and process samples to preserve phosphorylation status; use phosphatase inhibitors in lysis buffers to prevent dephosphorylation during extraction.

• Western blotting: Utilize freshly prepared samples whenever possible. Given that the predicted molecular weight of HSF1 is approximately 57-82 kDa , use 8-10% SDS-PAGE gels for optimal separation. Transfer to PVDF membranes at low current (30-40mA) overnight at 4°C to ensure complete transfer of higher molecular weight proteins.

• Immunofluorescence: Fix cells with 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100. Use a blocking solution containing 3-5% BSA to reduce background. Incubate with the primary antibody at dilutions between 1:100 and 1:200 .

• Storage and handling: Store the antibody at -20°C for long-term preservation, and at 4°C for short-term use. Avoid repeated freeze-thaw cycles as they can degrade antibody quality and specificity .

How can I validate the specificity of the Phospho-HSF1 (Ser307) Antibody in my experimental system?

To validate antibody specificity in your experimental system, implement these methodological approaches:

• Positive and negative controls: Use samples known to contain phosphorylated HSF1 at Ser307 (e.g., heat-shocked cells) as positive controls. For negative controls, treat samples with lambda phosphatase to remove phosphorylation.

• Phospho-knockout/knockdown validation: As demonstrated in research by Guettouche et al. (2005), compare antibody detection between wild-type HSF1 and S307A mutant forms where the phosphorylation site has been eliminated .

• Phosphorylation modulation: Treat cells with ERK pathway inhibitors (e.g., FR180204) which may enhance Ser307 phosphorylation, or with MEK inhibitors (e.g., U0126) which reportedly influence the phosphorylation pattern of HSF1 . Compare antibody detection before and after treatment.

• Peptide competition assay: Pre-incubate the antibody with excess phosphopeptide immunogen to block specific binding sites, which should eliminate or significantly reduce signal if the antibody is specific.

How does phosphorylation at Ser307 regulate HSF1 activity in stress response pathways?

Phosphorylation at Ser307 represents a key regulatory mechanism in HSF1 function. Research indicates that Ser307 phosphorylation acts as a repressive modification that inhibits HSF1 transcriptional activity . In the stress response pathway:

• Under non-stress conditions, HSF1 is maintained in an inactive monomeric form, partially through phosphorylation at sites including Ser307 .

• When cells are exposed to stressors like heat shock, HSF1 undergoes a complex series of post-translational modifications, including altered phosphorylation patterns.

• Studies using knock-in mouse models where Ser303 and Ser307 were replaced with alanine (Hsf1 303A/307A) demonstrated that elimination of these phosphorylation sites reduces the threshold of HSF1 activation, resulting in mild HSF1 activation even under non-stressed physiological conditions .

• This enhanced HSF1 activity in the absence of Ser307 phosphorylation leads to increased expression of heat shock proteins (HSPs) and altered cellular responses to stress .

The interplay between Ser307 phosphorylation and other post-translational modifications provides a sophisticated regulatory mechanism that fine-tunes the cellular stress response.

What signaling pathways are involved in regulating HSF1 Ser307 phosphorylation?

The regulation of HSF1 Ser307 phosphorylation involves several interconnected signaling pathways:

• MEK-ERK Pathway: Research has revealed complex interactions between MEK, ERK, and HSF1 phosphorylation. While ERK was initially reported to phosphorylate HSF1 at Ser307 , more recent studies suggest a more nuanced relationship:

  • MEK directly phosphorylates HSF1 at Ser326, which appears to repress Ser307 phosphorylation .

  • ERK inhibition can impact HSF1 Ser307 phosphorylation indirectly via MEK .

  • Activated ERK phosphorylates Thr292/386 to inhibit MEK1, creating a regulatory feedback loop .

• Stress Response Pathway: Heat shock and other stressors trigger changes in HSF1 phosphorylation status, including alterations in Ser307 phosphorylation .

These findings indicate that HSF1 Ser307 phosphorylation is regulated through complex crosstalk between multiple signaling pathways, allowing for fine-tuned control of the cellular stress response.

What is the relationship between Ser307 phosphorylation and other post-translational modifications of HSF1?

HSF1 undergoes multiple post-translational modifications that collectively regulate its activity in a coordinated manner:

• Multiple phosphorylation sites: Studies using mass spectrometry and sequencing have identified at least 12 serine residues that undergo phosphorylation in heat-activated human HSF1, including Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444 .

• Interplay between phosphorylation sites: Evidence suggests regulatory interactions between different phosphorylation sites:

  • Ser326 phosphorylation appears to contribute significantly to HSF1 activation by heat stress, while Ser307 phosphorylation has a repressive effect .

  • Phosphomimetic mutant HSF1 S326D proteins displayed reduced basal Ser307 phosphorylation and resisted induction of this phosphorylation by MEK inhibition, suggesting that Ser326 phosphorylation by MEK represses Ser307 phosphorylation .

• Other modifications: Beyond phosphorylation, HSF1 undergoes other post-translational modifications including acetylation, sumoylation, and ubiquitination at various residues . These modifications work in concert to create a complex regulatory network controlling HSF1 function.

The intricate interplay between these various modifications creates a sophisticated regulatory system that fine-tunes HSF1 activity in response to different cellular conditions and stressors.

How can Phospho-HSF1 (Ser307) Antibody be used to investigate the relationship between HSF1 activity and tumorigenesis?

The Phospho-HSF1 (Ser307) Antibody offers valuable tools for exploring the complex relationship between HSF1 activity and cancer development:

• Monitoring HSF1 activation status in tumors: Research has demonstrated that HSF1 is required for malignant transformation and essential for tumor cell survival . By using Phospho-HSF1 (Ser307) Antibody to assess phosphorylation status, researchers can evaluate the activation state of HSF1 in different tumor types.

• Investigating metabolic reprogramming: Studies with Hsf1 303A/307A mice showed that loss of HSF1 S303/S307 phosphorylation leads to age-associated obesity and obesity-related chronic inflammation, which are risk factors for cancer initiation and development . The antibody can be used to monitor changes in HSF1 phosphorylation during metabolic alterations associated with tumorigenesis.

• Evaluating drug resistance mechanisms: Research demonstrated that lack of HSF1 phosphorylation at S303/S307 significantly increased cell survival when cells were exposed to chemotherapeutic reagents . Phospho-HSF1 (Ser307) Antibody can be employed to investigate how changes in HSF1 phosphorylation status correlate with drug sensitivity and resistance in cancer cells.

• Assessing therapeutic interventions: Since HSF1 activation promotes tumor growth, therapies targeting HSF1 activity may have anti-cancer potential. The antibody can be used to monitor changes in Ser307 phosphorylation following treatment with pathway-specific inhibitors or novel therapeutic agents.

How might alterations in HSF1 Ser307 phosphorylation contribute to disease pathogenesis beyond cancer?

Research suggests that altered HSF1 Ser307 phosphorylation may contribute to multiple disease states beyond cancer:

• Metabolic disorders: The Hsf1 303A/307A mouse model demonstrated that loss of HSF1 S303/S307 phosphorylation leads to age-associated obesity and chronic inflammation , suggesting a role in metabolic regulation. Researchers can use the Phospho-HSF1 (Ser307) Antibody to investigate:

  • Changes in HSF1 phosphorylation status in obesity models

  • Correlations between HSF1 phosphorylation and insulin resistance

  • Alterations in HSF1 activity in tissues affected by metabolic syndrome

• Neurodegenerative diseases: The heat shock response and HSF1 activity are implicated in protein misfolding disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. The antibody can help examine:

  • Whether disease states correlate with alterations in HSF1 Ser307 phosphorylation

  • If changes in HSF1 regulation contribute to decreased proteostasis in neurodegenerative conditions

  • The potential of targeting HSF1 phosphorylation as a therapeutic approach

• Inflammatory disorders: Given the connection between HSF1 activity, inflammation, and cellular stress responses, researchers can employ the antibody to explore:

  • Changes in HSF1 Ser307 phosphorylation during acute and chronic inflammatory conditions

  • How alterations in HSF1 regulation might contribute to autoimmune diseases

  • The relationship between stress response pathways and inflammatory signaling

What experimental approaches can be used to manipulate HSF1 Ser307 phosphorylation for functional studies?

Several sophisticated experimental approaches can be employed to manipulate HSF1 Ser307 phosphorylation for functional studies:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated knock-in of S307A or S307D mutations to generate phospho-null or phospho-mimetic HSF1 variants

  • Conditional expression systems for temporally controlled expression of mutant HSF1

  • Generation of cell lines or animal models similar to the Hsf1 303A/307A knock-in mice

• Pharmacological interventions:

  • MEK inhibitors (e.g., U0126) and ERK inhibitors (e.g., FR180204, Sch772984) can be used to modulate the signaling pathways that influence HSF1 Ser307 phosphorylation

  • Selective kinase activators or inhibitors to target specific pathways involved in HSF1 regulation

• Combined approaches for temporal and spatial precision:

  • Optogenetic control of kinase activity to achieve temporal manipulation of HSF1 phosphorylation

  • Cell type-specific expression of HSF1 mutants to investigate tissue-specific effects

• Readout systems:

  • Reporter gene assays using HSF1-responsive promoters to monitor transcriptional activity

  • Chromatin immunoprecipitation (ChIP) with Phospho-HSF1 (Ser307) Antibody to assess DNA binding properties

  • Proteomics approaches to identify interactome changes associated with altered Ser307 phosphorylation status

What are common issues encountered when using Phospho-HSF1 (Ser307) Antibody and how can they be resolved?

Researchers may encounter several challenges when working with Phospho-HSF1 (Ser307) Antibody:

• Low signal intensity in Western blots:

  • Possible cause: Rapid dephosphorylation during sample preparation

  • Solution: Ensure samples are harvested rapidly and processed in the presence of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and β-glycerophosphate)

  • Alternative approach: Increase antibody concentration (up to 1:500 dilution) and extend incubation time to overnight at 4°C

• High background in immunofluorescence:

  • Possible cause: Insufficient blocking or non-specific binding

  • Solution: Increase blocking time (2-3 hours at room temperature) and concentration (5% BSA) before antibody incubation

  • Alternative approach: Include 0.1% Tween-20 in wash buffers and extend washing steps

• Inconsistent results across experiments:

  • Possible cause: Variable phosphorylation status due to cell culture conditions

  • Solution: Standardize cell culture conditions, including serum levels, cell density, and passage number

  • Alternative approach: Include positive controls (e.g., cells treated with known modulators of HSF1 phosphorylation) in each experiment

• Cross-reactivity concerns:

  • Possible cause: Antibody recognizing similar phosphorylation motifs in other proteins

  • Solution: Verify specificity using phospho-null HSF1 mutants (S307A) as negative controls

  • Alternative approach: Perform peptide competition assays to confirm specificity

How should results from Phospho-HSF1 (Ser307) Antibody experiments be interpreted in the context of HSF1 activation states?

Interpreting results from Phospho-HSF1 (Ser307) Antibody experiments requires careful consideration of HSF1's complex regulation:

• Understanding the relationship between phosphorylation and activity:

  • Ser307 phosphorylation generally correlates with repressed HSF1 transcriptional activity

  • Decreased Ser307 phosphorylation may indicate HSF1 activation, but this should be confirmed with additional markers

  • Consider that other phosphorylation events (e.g., Ser326) may have dominant effects on HSF1 activity

• Contextual interpretation across experimental conditions:

  • Stress conditions: Heat shock typically alters HSF1 phosphorylation patterns, including at Ser307

  • Pathway modulation: Changes in Ser307 phosphorylation following treatment with inhibitors (e.g., U0126, FR180204) should be interpreted in the context of pathway interactions

  • Temporal dynamics: Consider the kinetics of phosphorylation changes, as early and late events may have different functional significance

• Correlation with functional readouts:

  • Heat shock protein expression (HSP70, HSP90, HSP25) should be measured as downstream indicators of HSF1 activity

  • HSF1 trimerization and nuclear localization status provide additional information about activation state

  • DNA binding capacity (assessed by ChIP or EMSA) offers direct measurement of HSF1 functional activity

• Integrating multiple phosphorylation sites:

  • When possible, assess multiple HSF1 phosphorylation sites simultaneously (e.g., Ser303, Ser307, Ser326)

  • Consider that certain phosphorylation events may be interdependent or have hierarchical relationships

  • Recognize that the net effect on HSF1 activity results from the integration of multiple modifications

What complementary techniques should be used alongside Phospho-HSF1 (Ser307) Antibody to comprehensively assess HSF1 activity?

A multi-method approach provides the most comprehensive assessment of HSF1 activity:

• Assessing HSF1 phosphorylation status:

  • Use antibodies against multiple phosphorylation sites (Ser303, Ser307, Ser326) to create a phosphorylation profile

  • Employ Phos-tag SDS-PAGE to separate differently phosphorylated forms of HSF1

  • Consider mass spectrometry for unbiased identification of all phosphorylation sites

• Evaluating HSF1 transcriptional activity:

  • Measure expression of HSF1 target genes (HSP70, HSP90, HSP25) using qRT-PCR or Western blotting

  • Utilize reporter gene assays with heat shock element (HSE)-containing promoters

  • Perform RNA-seq to assess genome-wide transcriptional changes

• Analyzing HSF1 protein interaction and localization:

  • Examine HSF1 trimerization using non-denaturing gel electrophoresis

  • Assess nuclear translocation through subcellular fractionation or immunofluorescence microscopy

  • Investigate HSF1 interactions with regulators (e.g., HSP90) via co-immunoprecipitation

• Studying HSF1 chromatin binding:

  • Perform ChIP assays using total HSF1 antibodies alongside phospho-specific antibodies

  • Utilize ChIP-seq for genome-wide analysis of HSF1 binding sites

  • Consider CUT&RUN or CUT&Tag for higher resolution analysis of HSF1 genomic occupancy

By integrating data from these complementary approaches, researchers can develop a comprehensive understanding of how Ser307 phosphorylation influences HSF1 function in different biological contexts.