Phospho-HSPB1 (S82) Recombinant Monoclonal Antibody

What is HSPB1 and what function does phosphorylation at S82 serve?

HSPB1 (also known as HSP27, HSP28) is an abundant molecular chaperone primarily found in striated muscle tissue. It functions as a critical molecular chaperone that maintains denatured proteins in a folding-competent state, preventing deleterious aggregation during cellular stress conditions . The phosphorylation of HSPB1 at serine 82 (S82) represents one of three major phosphorylation sites (along with S15 and S78/S86) in the N-terminal region of the protein, outside the alpha-crystallin domain . This phosphorylation is catalyzed by MAPKAPK2/3 (mitogen-activated protein kinase-activated protein kinase 2/3) in response to various stimuli, particularly mechanical stress . When HSPB1 becomes phosphorylated at S82, it undergoes significant conformational changes that drive its translocation to Z-discs and locations with elevated traction force within the cytoskeleton, where it interacts with mechanosensitive clients such as FLNC . This phosphorylation-induced spatial reorganization is crucial for HSPB1's role in stress resistance and actin cytoskeletal organization .

How does the quaternary structure of HSPB1 change following S82 phosphorylation?

Phosphorylation at S82 dramatically alters the quaternary structure of HSPB1, inducing a transition from large oligomeric complexes to smaller species. Research has demonstrated that even low levels of phosphorylation (0.3-0.4 mole phosphate per mole of protein) can initiate significant reductions in the quantity of large oligomeric species while increasing the fraction of smaller oligomers . When phosphorylation reaches approximately 0.9-1.0 mole phosphate per mole of protein or higher, complete dissociation of large oligomers into smaller species occurs . These resulting species typically exhibit an apparent molecular mass of 85-90 kDa for wild-type HSPB1 . The oligomeric state transformation is critical because it dictates HSPB1's chaperone activity, substrate binding capacity, and cellular distribution, all of which influence its ability to protect against cellular stresses and regulate cytoskeletal dynamics .

What are the validated applications for Phospho-HSPB1 (S82) Recombinant Monoclonal Antibody?

The Phospho-HSPB1 (S82) Recombinant Monoclonal Antibody has been validated for multiple experimental applications:

The antibody specifically targets the human HSPB1 phosphorylated at the Ser82 residue and belongs to the rabbit IgG class . It has been purified using affinity-chromatography methods, ensuring high specificity for the phosphorylated form of the protein . Most experimental validations have been performed with human samples, though cross-reactivity with other species may be possible based on sequence homology .

How can I distinguish between phosphorylation-dependent and independent functions of HSPB1 in mechanotransduction studies?

To differentiate between phosphorylation-dependent and independent functions of HSPB1 in mechanotransduction research, a multi-faceted experimental approach is recommended:

• Phosphomutant expression systems: Generate cell lines expressing phosphomimetic (S15,86E) and non-phosphorylatable (S15,86A) HSPB1 mutants in HSPB1-null backgrounds. Research has demonstrated that only phosphorylatable HSPB1, which displays mechanoaccumulation to tensed actin stress fibers, can restore activities lost in HSPB1-null cells, including actin remodeling, cell spreading, and cell motility .

• Mechanical stimulation assays: Subject cells to uniaxial cyclic stretch stimulation and quantify the phosphorylation increase (typically 1.9-fold) using phospho-specific antibodies against S82 . Compare the subcellular distribution of wild-type, S15,86A, and S15,86E HSPB1 variants using immunofluorescence imaging after mechanical stimulation.

• Pharmacological inhibition: Employ MK2 inhibitors to block HSPB1 phosphorylation while maintaining HSPB1 expression, then assess whether mechanical stimulus-dependent responses are inhibited.

• Subcellular localization analysis: Analyze intensity line profiles to differentiate between phosphorylated HSPB1 accumulation patterns (which typically show lines slightly displaced from focal adhesions) versus non-phosphorylatable variants that fail to accumulate at these sites .

This comprehensive approach enables researchers to attribute specific cellular functions to the phosphorylation state of HSPB1 rather than merely to the presence of the protein itself.

What are the methodological considerations when investigating the effect of HSPB1 S82 phosphorylation on protein-protein interactions in mechanical stress models?

When studying how S82 phosphorylation affects HSPB1's protein-protein interactions during mechanical stress, several methodological considerations are essential:

• Experimental environment control: Cellular crowding significantly impacts HSPB1 oligomerization. Research using crowding reagents like TMAO has shown that HSPB1 forms exclusively large oligomers under crowded conditions that mimic the cellular environment (>200 mg/ml total protein concentration) . Therefore, in vitro experiments should account for molecular crowding effects.

• Phosphorylation level quantification: Precise quantification of phosphorylation stoichiometry is critical. Different phosphorylation levels (0.3-0.4 versus 0.9-1.0 mole phosphate per mole of protein) drive significantly different oligomeric states , which directly impact interaction capabilities.

• Tag interference assessment: When investigating protein-protein interactions, consider that even small tags can alter HSPB1 quaternary structure. Studies have demonstrated that N-terminal His tags affect HSPB1 structure . Design experiments with appropriate controls for tagged constructs.

• Heterooligomer formation analysis: HSPB1 forms heterooligomeric complexes with distinct physicochemical properties compared to homooligomers. Methodologies should account for potential interactions with other small heat shock proteins that might mask or enhance the effects of S82 phosphorylation .

• Cross-linking studies: Implement chemical cross-linking followed by mass spectrometry to capture transient interactions that might be missed in standard co-immunoprecipitation approaches, especially in the context of mechanically stressed cells where phosphorylation status changes dynamically.

These considerations help avoid methodological artifacts and ensure more physiologically relevant insights into HSPB1's interactome as regulated by S82 phosphorylation.

How does the integration of mechanical signaling and HSPB1 phosphorylation contribute to cytoskeletal remodeling in disease models?

The integration of mechanical signaling and HSPB1 phosphorylation plays a crucial role in cytoskeletal remodeling, with significant implications for disease models, particularly muscle myopathies . This complex relationship involves:

• Mechanotransduction pathway activation: Mechanical stress activates the p38 MAPK signaling cascade, leading to MAPKAPK-2 activation and subsequent HSPB1 phosphorylation . This phosphorylation represents a critical molecular switch that determines HSPB1's subcellular localization and function.

• Cytoskeletal tension regulation: Phosphorylated HSPB1 translocates to Z-discs and regions of elevated traction force within the cytoskeleton . This mechanical localization allows phospho-HSPB1 to interact with key cytoskeletal components like FLNC that are critical for maintaining cellular integrity under mechanical stress.

• Protein quality control: When normal mechanical forces become excessive, proteins can unfold and form deleterious aggregates that overwhelm the protein quality control network . HSPB1 phosphorylation modulates its chaperone activity to prevent these pathological protein deposits that underlie various muscle myopathies .

• Cell behavioral outcomes: Research using CRISPR/Cas9-edited HSPB1-null cells has demonstrated that phosphorylation-deficient HSPB1 cannot restore normal actin cytoskeletal remodeling, cell spreading, or cell motility following mechanical stimulation . These altered cellular behaviors provide potential mechanisms for disease progression.

These integrated processes suggest that dysregulation of HSPB1 phosphorylation in response to mechanical cues may contribute to disease pathogenesis by disrupting cytoskeletal homeostasis, which could be targeted therapeutically in various myopathies.

What are the optimal conditions for using Phospho-HSPB1 (S82) Recombinant Monoclonal Antibody in Western blot analysis?

For optimal Western blot analysis using Phospho-HSPB1 (S82) Recombinant Monoclonal Antibody, the following methodological approach is recommended:

• Sample preparation:

  • Extract proteins using a lysis buffer containing phosphatase inhibitors to preserve phosphorylation status

  • Include positive controls (cells treated with stress inducers that activate the p38 MAPK pathway) and negative controls (phosphatase-treated samples)

• Dilution optimization:

  • Start with a 1:1000 dilution (within the recommended 1:500-1:5000 range)

  • For low abundance samples, use higher antibody concentrations (1:500)

  • For high expression systems, use more dilute antibody (1:2000-1:5000)

• Detection system compatibility:

  • The antibody works effectively with both chemiluminescent and fluorescent secondary detection systems

  • For quantitative analysis, fluorescent detection provides better linearity

• Signal verification:

  • Validate signal specificity using HSPB1 knockout cells or MAPKAPK-2 inhibitor-treated cells

  • Compare phosphorylated and total HSPB1 levels using paired blots or stripping and reprobing

• Molecular weight reference:

  • Look for a band at approximately 27 kDa, corresponding to phosphorylated HSPB1

  • Be aware that phosphorylation may cause a slight mobility shift compared to non-phosphorylated HSPB1

This methodological approach ensures reliable and reproducible detection of phosphorylated HSPB1 at the S82 site, critical for studies investigating mechanical stress responses and cytoskeletal dynamics.

How can I design experiments to study the dynamics of HSPB1 phosphorylation in response to mechanical stress?

To effectively study HSPB1 phosphorylation dynamics in response to mechanical stress, consider this comprehensive experimental design approach:

• Mechanical stimulation setup:

  • Implement a uniaxial cyclic stretch system to apply controlled mechanical stress to cells

  • Use flexible-bottomed culture plates with varying strain magnitudes (5-20%) and frequencies (0.5-2 Hz)

  • Include unstimulated controls to establish baseline phosphorylation levels

• Temporal analysis:

  • Collect samples at multiple timepoints (5, 15, 30, 60 minutes, 2, 4, 8 hours) after stretch initiation

  • This allows capturing the rapid initial phosphorylation and potential subsequent dephosphorylation or adaptive responses

• Phosphorylation quantification:

  • Use Western blotting with Phospho-HSPB1 (S82) antibody at 1:1000 dilution

  • Normalize to total HSPB1 levels using a non-phospho-specific antibody

  • Consider flow cytometry for single-cell resolution of phosphorylation heterogeneity

• Subcellular localization assessment:

  • Perform immunofluorescence imaging using the Phospho-HSPB1 (S82) antibody (1:100 dilution)

  • Co-stain for cytoskeletal markers (actin, α-actinin for Z-discs)

  • Use intensity line profile analysis to quantify phospho-HSPB1 recruitment to cytoskeletal structures

• Signaling pathway integration:

  • Include inhibitors of upstream kinases (p38 MAPK inhibitors, MAPKAPK-2 inhibitors)

  • Assess the impact of these inhibitors on both HSPB1 phosphorylation and downstream cellular responses

  • This approach differentiates direct mechanical effects from secondary signaling consequences

• Functional outcome correlation:

  • Simultaneously assess cytoskeletal remodeling, cell spreading, and motility

  • Correlate these outcomes with phosphorylation dynamics to establish causative relationships

This experimental design enables comprehensive characterization of how mechanical forces regulate HSPB1 phosphorylation and the resulting functional consequences.

What controls should be included when validating the specificity of Phospho-HSPB1 (S82) antibody in immunohistochemistry applications?

When validating the specificity of Phospho-HSPB1 (S82) antibody for immunohistochemistry (IHC), a robust set of controls is essential to ensure accurate and reproducible results:

• Genetic controls:

  • HSPB1 knockout tissues/cells: Should show complete absence of staining

  • CRISPR/Cas9-edited cells expressing non-phosphorylatable HSPB1 (S82A mutant): Should show minimal or no staining even under conditions that normally induce phosphorylation

  • Phosphomimetic HSPB1 (S82E) expressing cells: Should show constitutive staining

• Pharmacological controls:

  • Tissues/cells treated with p38 MAPK or MAPKAPK-2 inhibitors: Should show reduced phospho-S82 HSPB1 staining

  • Tissues/cells stimulated with stress inducers (heat shock, oxidative stress, mechanical strain): Should show enhanced phospho-S82 HSPB1 staining

  • Lambda phosphatase-treated sections: Should eliminate specific staining

• Antibody validation controls:

  • Antibody dilution series (starting from 1:50 to 1:200 as recommended) to determine optimal signal-to-noise ratio

  • Peptide competition assay using phospho-S82 peptide versus non-phosphorylated peptide

  • Comparison with alternative phospho-S82 HSPB1 antibodies from different suppliers or clones

• Technical controls:

  • Omission of primary antibody to assess secondary antibody non-specific binding

  • Isotype control at equivalent concentration to assess Fc receptor-mediated binding

  • Serial sections stained with total HSPB1 antibody to compare distribution patterns

• Cross-reactivity assessment:

  • Western blot validation of tissues before IHC to confirm single band at expected molecular weight

  • Multiple tissue types (including those with known high and low HSPB1 expression)

  • Testing across species if cross-reactivity is claimed

These comprehensive controls ensure that observed staining patterns truly represent phosphorylated HSPB1 at S82, critical for accurate interpretation of results in studies of mechanotransduction and stress response pathways.

How do I interpret discrepancies between in vitro and cellular studies of HSPB1 phosphorylation effects on oligomerization?

Interpreting discrepancies between in vitro and cellular studies of HSPB1 phosphorylation effects requires understanding several key factors that influence experimental outcomes:

• Molecular crowding effects: In vitro studies often occur in dilute solutions, whereas cells maintain protein concentrations exceeding 200 mg/ml. Research has demonstrated that crowding reagents like TMAO cause HSPB1 mutants to form exclusively large oligomers, even at concentrations that would lead to dissociation in normal aqueous conditions . When interpreting contradictory results, evaluate whether the in vitro conditions adequately mimic cellular crowding.

• Protein tagging artifacts: Previous cellular investigations have shown that even small N-terminal His tags affect the quaternary structure of HSPB1 . When comparing studies, examine whether the HSPB1 constructs include tags (V5, FLAG, GFP, etc.) that might artificially alter oligomerization behavior independent of phosphorylation status.

• Interacting partner proteins: In cellular lysates, dissociated HSPB1 species can interact with partner proteins, stabilizing small molecular mass entities that are never detected in purified protein systems . Analysis should consider whether observed differences might result from protein-protein interactions present only in the cellular context.

• Phosphorylation heterogeneity: Unlike in vitro systems where phosphorylation can be precisely controlled, cellular HSPB1 often exists with heterogeneous phosphorylation at multiple sites (S15, S78, S82), creating mixed populations of oligomers . This heterogeneity complicates direct comparisons with defined in vitro states.

• Methodological resolution limits: Techniques used to assess oligomerization (SEC, native PAGE, cross-linking) have different resolution capabilities and may detect different subpopulations of oligomers. Consider whether methodological differences, rather than biological differences, explain discrepant results.

To address these discrepancies, design experiments that bridge in vitro and cellular contexts, such as introducing crowding agents to in vitro systems or using phosphosite-specific antibodies to isolate distinct phosphorylated populations from cells.

What are the implications of HSPB1 phosphorylation status for interpreting experimental results in cytoskeletal dynamics studies?

The phosphorylation status of HSPB1 has profound implications for interpreting experimental results in cytoskeletal dynamics studies, requiring careful consideration of several factors:

• Baseline phosphorylation variability: In unstimulated fibroblasts, HSPB1 exhibits a baseline phosphorylation level that can vary significantly between cell lines and culture conditions. This baseline increases approximately 1.9-fold upon uniaxial cyclic stretch stimulation . Experimental interpretations should account for this variable starting point when comparing different cell models or treatment conditions.

• Spatial redistribution effects: Phosphorylated HSPB1 undergoes significant spatial redistribution, accumulating at Z-discs and sites of elevated traction force within the cytoskeleton . This means that total cellular phospho-HSPB1 levels may remain unchanged while its functional impact shifts dramatically due to altered localization. Analysis should incorporate subcellular localization data alongside phosphorylation quantification.

• Functional restoration thresholds: Research using HSPB1-null cells has demonstrated that only phosphorylatable HSPB1 can restore normal cytoskeletal activities like actin remodeling, cell spreading, and motility . This suggests a threshold effect where partial phosphorylation may be insufficient for normal function. Quantitative correlations between phosphorylation levels and functional outcomes provide critical context for interpretation.

• Temporal dynamics considerations: The timeline of HSPB1 phosphorylation relative to observed cytoskeletal changes is crucial. Some studies may capture early phosphorylation events before functional consequences manifest, while others may observe functional changes after phosphorylation levels have normalized. Time-course experiments are essential for establishing cause-effect relationships.

• Mechanosensor coordination: HSPB1 functions within a complex network of mechanosensitive proteins. Its phosphorylation may be a consequence rather than initiator of cytoskeletal changes in some contexts. Experimental designs that selectively manipulate HSPB1 phosphorylation while maintaining other mechanosensing pathways help differentiate its specific contribution.

These considerations highlight the need for integrated analysis of HSPB1 phosphorylation dynamics, localization patterns, and functional outcomes when interpreting cytoskeletal research results.

How can phosphorylation-specific antibodies like Phospho-HSPB1 (S82) help distinguish between different stress response pathways?

Phosphorylation-specific antibodies like Phospho-HSPB1 (S82) serve as powerful tools for distinguishing between different stress response pathways through several methodological approaches:

• Pathway-specific activation patterns: Different stressors activate distinct upstream kinase cascades that converge on HSPB1 phosphorylation. For example, mechanical stress primarily activates p38 MAPK pathway leading to S82 phosphorylation , while other stressors may preferentially activate different sites. By using phospho-site specific antibodies in combination (S15, S78, and S82), researchers can create "phosphorylation signatures" characteristic of specific stress pathways.

• Phosphorylation kinetics analysis: The temporal profile of S82 phosphorylation differs between stress types. Mechanical stress typically induces rapid phosphorylation (detectable within 5-15 minutes) , while other stressors may show delayed or prolonged phosphorylation patterns. Time-course studies using Phospho-HSPB1 (S82) antibody can differentiate these responses.

• Subcellular localization mapping: Different stress pathways direct phosphorylated HSPB1 to distinct subcellular compartments. Mechanically induced phospho-HSPB1 targets Z-discs and regions of cytoskeletal tension , while heat shock may direct it toward protein aggregates. Immunofluorescence studies using the Phospho-HSPB1 (S82) antibody can map these location-specific responses.

• Inhibitor profiling strategy: By combining Phospho-HSPB1 (S82) antibody detection with selective pathway inhibitors (p38 MAPK inhibitors, JNK inhibitors, PKC inhibitors), researchers can determine which upstream pathways contribute to S82 phosphorylation under different stress conditions. This approach creates inhibitor sensitivity profiles characteristic of specific pathways.

• Client protein interaction analysis: Phosphorylated HSPB1 interacts with different client proteins depending on the activating stress. Combining Phospho-HSPB1 (S82) antibody with proximity ligation assays or co-immunoprecipitation allows mapping of stress-specific interactomes.

This multi-faceted methodological approach using phospho-specific antibodies enables researchers to decipher the complex language of cellular stress responses with high precision and develop targeted interventions for stress-related pathologies.

How does the function of phosphorylated HSPB1 at S82 compare with phosphorylation at other sites (S15, S78)?

The functional consequences of HSPB1 phosphorylation exhibit distinct patterns depending on which serine residue is modified, with important implications for experimental design and interpretation:

While all three sites are phosphorylated by MAPKAPK2/3 in response to stress stimuli, S82 phosphorylation appears to play a particularly critical role in oligomer dissociation and mechanosensitive functions . Research has shown that phosphorylation at a single site can induce preliminary dissociation of large oligomers, but complete dissociation typically requires phosphorylation at multiple sites .

The localization pattern of phospho-S82 HSPB1 is more precisely defined, with clear accumulation at Z-discs and regions of cytoskeletal tension following mechanical stimulation . This specific localization pattern likely explains why S82 phosphorylation is particularly important for HSPB1's functions in cytoskeletal remodeling and cell motility .

Studies with phosphomimetic mutations further reveal that S82 phosphorylation cannot be functionally replaced by modifications at other sites, suggesting unique structural consequences or interaction partners specific to this phosphorylation event . This site-specific functionality should be considered when designing experiments targeting HSPB1 phosphorylation in different physiological contexts.

What differences exist between phospho-specific and total HSPB1 antibodies in research applications?

Phospho-specific HSPB1 antibodies (like Phospho-HSPB1 (S82)) and total HSPB1 antibodies serve complementary roles in research, with distinct advantages and limitations:

The choice between these antibody types depends on the research question. Phospho-specific antibodies excel in signaling studies, stress response analysis, and mechanotransduction research where the activation state of HSPB1 is crucial . They provide temporal resolution of pathway activation and spatial information about where active HSPB1 localizes within the cell .

The most robust experimental approach combines both antibody types, using total HSPB1 antibodies to normalize phospho-specific signals and provide context for interpreting phosphorylation changes, particularly in comparative studies across different conditions or cell types.

How can Phospho-HSPB1 (S82) antibodies contribute to understanding pathological protein aggregation in muscle myopathies?

Phospho-HSPB1 (S82) antibodies offer valuable insights into the mechanisms underlying pathological protein aggregation in muscle myopathies through several research applications:

• Aggregate composition analysis: In muscle biopsies from myopathy patients, Phospho-HSPB1 (S82) antibodies can determine whether phosphorylated HSPB1 is sequestered within protein aggregates. This helps distinguish between aggregates where HSPB1 is a constituent versus those where it is actively engaged in attempted disaggregation . Immunohistochemistry using 1:50-1:200 dilutions provides optimal visualization of aggregate composition .

• Phosphorylation status mapping: The balance between phosphorylated and non-phosphorylated HSPB1 is critical for its chaperone function. Excessive force can lead to protein unfolding and formation of deleterious aggregates that overwhelm the protein quality control network . Quantitative comparison of phospho-HSPB1 to total HSPB1 ratios in healthy versus diseased muscle identifies dysregulation of this protective mechanism.

• Mechanical stress response evaluation: Many myopathies involve defective mechanotransduction. Since HSPB1 phosphorylation occurs in response to mechanical stress , reduced phospho-HSPB1 levels despite mechanical stimulation could indicate pathway defects contributing to disease pathogenesis. Ex vivo stretch experiments on muscle biopsies followed by phospho-HSPB1 detection can reveal these abnormalities.

• Therapeutic intervention monitoring: Potential therapies targeting the protein quality control network can be assessed by monitoring changes in HSPB1 phosphorylation status. Restoration of normal phospho-HSPB1 levels and distribution patterns may serve as biomarkers for treatment efficacy.

• Client protein interaction mapping: Phosphorylated HSPB1 interacts with specific client proteins like FLNC at sites of mechanical stress . In myopathies, these interactions may be disrupted. Co-immunoprecipitation using Phospho-HSPB1 (S82) antibodies followed by mass spectrometry can identify altered interaction networks contributing to disease.

These applications demonstrate how Phospho-HSPB1 (S82) antibodies serve not only as diagnostic tools but also as instruments for mechanistic understanding and therapeutic development in muscle myopathies characterized by pathological protein aggregation.

What are common challenges in detecting phosphorylated HSPB1 and how can they be overcome?

Detecting phosphorylated HSPB1 presents several technical challenges that can be systematically addressed through optimized methodologies:

• Rapid dephosphorylation during sample processing

  • Challenge: Phosphorylated HSPB1 can be rapidly dephosphorylated by endogenous phosphatases during sample collection and processing.

  • Solution: Immediately lyse samples in buffer containing phosphatase inhibitor cocktails. Keep samples cold (4°C) throughout processing and add EDTA (5mM) to chelate cations required for phosphatase activity.

• Low signal-to-noise ratio in minimally phosphorylated samples

  • Challenge: Baseline phosphorylation levels may be too low for reliable detection.

  • Solution: Implement signal amplification methods such as TSA (tyramide signal amplification) for IHC applications. For Western blotting, use more concentrated antibody dilutions (1:500) and higher protein loading (30-50μg per lane) .

• Non-specific binding with other phosphorylated proteins

  • Challenge: Some phospho-antibodies may cross-react with similarly phosphorylated motifs in other proteins.

  • Solution: Validate specificity using HSPB1 knockout samples as negative controls. Perform peptide competition assays with phosphorylated and non-phosphorylated peptides to confirm binding specificity.

• Heterogeneous phosphorylation across HSPB1 population

  • Challenge: HSPB1 may be simultaneously phosphorylated at multiple sites (S15, S78, S82), creating a mixed population difficult to quantify.

  • Solution: Use site-specific phospho-antibodies in parallel analyses. Consider Phos-tag™ SDS-PAGE to separate different phosphorylated species based on phosphorylation state.

• Antibody batch variation affecting reproducibility

  • Challenge: Different antibody lots may have varying sensitivity and specificity.

  • Solution: Maintain consistent positive controls across experiments. Consider creating standard curves with recombinant phosphorylated protein to normalize between antibody batches.

• Fixation-induced epitope masking in tissue samples

  • Challenge: Formalin fixation can mask the phospho-epitope in IHC applications.

  • Solution: Optimize antigen retrieval methods (try both heat-mediated and enzymatic approaches). For IHC, test different fixatives beyond formalin, such as periodate-lysine-paraformaldehyde, which better preserves phospho-epitopes.

These methodological refinements significantly improve the reliability and sensitivity of phosphorylated HSPB1 detection across various experimental applications.