Phospho-ROCK2 (Tyr722) Antibody

What is the specificity of Phospho-ROCK2 (Tyr722) antibody?

The Phospho-ROCK2 (Tyr722) antibody specifically detects endogenous levels of ROCK2 protein only when phosphorylated at Tyrosine 722. This high specificity is achieved through a careful purification process where antibodies are produced by immunizing rabbits with synthetic phosphopeptide and KLH conjugates, then purified using affinity chromatography with epitope-specific phosphopeptide. Importantly, non-phospho specific antibodies are removed through chromatography using non-phosphopeptide, ensuring the antibody only recognizes the phosphorylated form . When designing experiments requiring precise detection of phosphorylated ROCK2, this specificity is crucial for distinguishing between the phosphorylated and non-phosphorylated forms of the protein.

How should researchers prepare and store Phospho-ROCK2 (Tyr722) antibody for optimal performance?

For optimal antibody performance, researchers should adhere to specific storage and handling protocols. The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide at a concentration of 1.0mg/ml . Long-term storage should be maintained at -20°C, where the antibody remains stable for approximately one year. When designing experiments, researchers should avoid repeated freeze-thaw cycles which can degrade antibody quality. For Western blot applications, the standard working dilution should be optimized for each experimental system, though manufacturer recommendations typically provide a starting point. Always validate the antibody's performance in your specific experimental conditions before proceeding with critical experiments.

What experimental models are appropriate for Phospho-ROCK2 (Tyr722) antibody research?

The Phospho-ROCK2 (Tyr722) antibody demonstrates species reactivity with human, mouse, and rat samples (abbreviated as Hu Ms Rt) . This makes it suitable for various experimental models, including cultured cell lines and primary cells derived from these species. In published research, the antibody has been successfully employed in rat hippocampal neurons (RHNs) to study phosphorylation events . When designing experiments, researchers should consider that while the antibody has confirmed reactivity with these species, validation in specific cell types or tissues is recommended before extensive experimental work. For neurological research, rat hippocampal neurons have proven to be an effective model system for studying ROCK2 phosphorylation in the context of hypoxia-reoxygenation injury protection .

How does hydrogen sulfide (H₂S) influence ROCK2 phosphorylation at Tyr722, and what methodologies are optimal for investigating this relationship?

H₂S significantly promotes the phosphorylation of ROCK2 at Tyr722 while simultaneously inhibiting ROCK2 protein expression and activity in rat hippocampal neurons (RHNs). To investigate this relationship, researchers should employ a multi-faceted approach:

• Dose-response experiments: Treat RHNs with varying concentrations of NaHS (50, 100, 200 μmol/L) as an H₂S donor .

• Western blot analysis: Quantify both total ROCK2 protein expression and p-ROCK2 Y722 levels using specific antibodies .

• Activity assays: Measure ROCK2 kinase activity following H₂S treatment to correlate phosphorylation with functional changes .

• Mutational analysis: Utilize site-directed mutagenesis to generate ROCK2 Y722F (tyrosine to phenylalanine) mutants that cannot be phosphorylated at position 722, allowing for direct comparison with wild-type ROCK2 .

• Transfection experiments: Use recombinant plasmids (ROCK2 Y722-pEGFP-N1 and ROCK2 Y722F-pEGFP-N1) to examine the effects of exogenous wild-type and mutant ROCK2 expression on H₂S-induced phosphorylation .

Research has demonstrated that NaHS at concentrations of 50, 100, and 200 μmol/L significantly inhibited ROCK2 protein expression (p < 0.01) while promoting phosphorylation at Tyr722 (p < 0.05, 0.01, 0.01) in RHNs . These methodological approaches allow researchers to comprehensively analyze how H₂S regulates ROCK2 through Tyr722 phosphorylation.

What are the technical considerations for performing in vitro phosphorylation assays to study ROCK2 Tyr722 phosphorylation?

When designing in vitro phosphorylation assays for studying ROCK2 Tyr722 phosphorylation, researchers should consider the following technical protocols and parameters:

• Protein preparation: Prokaryotic expression systems can be used to produce recombinant ROCK2, though challenges exist with high molecular weight proteins. GST-tagged ROCK2 wild-type and Y722F mutant proteins should be generated using appropriate expression vectors (e.g., pGEX-6P-1) .

• Reaction components: The standard reaction mixture should contain:

  • 15 μL protein specimen

  • 100 μmol/L ATP

  • 100 ng Src kinase (with or without 100 μmol/L NaHS)

  • Kinase buffer (20 mM Tris-Cl, 100 mM KCl, 2 mM EGTA, 5 mM MgCl₂, pH 7.4)

• Reaction conditions: The mixture should be incubated with gentle shaking for 30 minutes at 37°C .

• Reaction termination: Add SDS-PAGE loading buffer to stop the phosphorylation reaction .

• Detection method: Use 8% SDS-PAGE for protein separation, followed by transfer to PVDF membranes. Block with 20% fetal bovine serum for 30 minutes, then incubate with anti-ROCK2 (phospho Y722) antibody overnight at 4°C. Visualize using appropriate secondary antibody and ECL-Plus reagent .

• Controls: Include reactions without Src kinase, without NaHS, and using the Y722F mutant form of ROCK2 that cannot be phosphorylated at position 722 .

These methodological details ensure reliable and reproducible results when investigating ROCK2 phosphorylation at Tyr722 in vitro.

What is the functional significance of ROCK2 Tyr722 phosphorylation in neuroprotection against hypoxia-reoxygenation injury?

ROCK2 Tyr722 phosphorylation plays a critical role in neuroprotection against hypoxia-reoxygenation (H/R) injury through several key mechanisms:

• ROCK2 activity inhibition: Phosphorylation at Tyr722 significantly reduces ROCK2 kinase activity, which is crucial as hyperactivation of the RhoA-ROCK signaling pathway exacerbates neuronal damage during ischemic events .

• Experimental evidence: Transfection with ROCK2 Y722-pEGFP-N1 plasmid significantly enhanced the protective effect of H₂S (NaHS) against H/R injury in rat hippocampal neurons, as measured by:

  • Increased cell viability

  • Reduced lactate dehydrogenase (LDH) release

  • Decreased neuron-specific enolase (NSE) release

• BKCa channel activation: ROCK2 Tyr722 phosphorylation mediates the H₂S-increased current of big-conductance Ca²⁺-activated K⁺ (BKCa) channels in neurons. This was demonstrated using whole-cell patch-clamp recording techniques, where NaHS (100 μmol/L) significantly increased BKCa current in GFP-ROCK2 Y722 transfected neurons compared to controls .

• Mechanistic pathway: The neuroprotective effect occurs through a sequential process where H₂S promotes Tyr722 phosphorylation → inhibits ROCK2 activity → opens BKCa channels → protects neurons from H/R injury .

The experimental approach to studying this pathway involves comparing wild-type ROCK2 with Y722F mutants that cannot be phosphorylated, demonstrating that the Y722F mutation significantly weakens the protective effects of H₂S against H/R injury .

How do ROCK2 interactions with NADPH oxidase components influence reactive oxygen species production in immune cells?

ROCK2 plays a significant role in regulating reactive oxygen species (ROS) production in monocytes through specific interactions with NADPH oxidase components:

• Protein-protein interactions: ROCK2 associates directly with p22phox, a membrane component of the NADPH oxidase complex, in human monocytes .

• Phosphorylation targets: Despite interacting with p22phox, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox/NOX2 directly. Instead, ROCK2 specifically phosphorylates p47phox at multiple serine residues (Ser304, Ser315, Ser320, and Ser328) .

• Functional consequences: The phosphorylation of p47phox by ROCK2 is critical for NADPH oxidase activation and subsequent ROS production in monocytes .

• Experimental validation:

  • In vitro kinase assays with recombinant proteins confirm that ROCK2 directly phosphorylates p47phox but not other NADPH oxidase components

  • Selective inhibition of ROCK2 activity and expression reduces both ROS production and p47phox phosphorylation in human monocytes

• Significance: This regulatory mechanism represents a potential therapeutic target for controlling excessive ROS production in inflammatory conditions .

The methodological approach to study these interactions involves both in vitro reconstitution experiments with purified components and cellular studies using selective ROCK2 inhibitors, providing complementary evidence for the role of ROCK2 in regulating NADPH oxidase activity.

What are the methodological approaches for analyzing ROCK2 Y722 phosphorylation in transfected cells?

When analyzing ROCK2 Y722 phosphorylation in transfected cells, researchers should implement a systematic methodology:

• Plasmid construction and transfection:

  • Design expression vectors containing either wild-type ROCK2 (ROCK2 Y722-pEGFP-N1) or mutant ROCK2 (ROCK2 Y722F-pEGFP-N1) fused to GFP

  • Include appropriate empty vector controls

  • Optimize transfection conditions for target cells (e.g., rat hippocampal neurons show highest transfection efficiency at 72 hours)

• Phosphorylation induction:

  • Treat transfected cells with phosphorylation inducers (e.g., NaHS at concentrations of 50, 100, 200 μmol/L)

  • Include appropriate vehicle controls

• Protein detection and quantification:

  • Perform Western blot analysis using antibodies against:

    • Total ROCK2

    • Phospho-ROCK2 (Tyr722)

    • GFP (to detect recombinant proteins)

  • Calculate phosphorylation ratios (p-ROCK2 Y722/ROCK2 and GFP-p-ROCK2 Y722/GFP-ROCK2 Y722)

• Functional assays:

  • Measure ROCK2 activity using kinase assays

  • Assess cellular outcomes (e.g., cell viability, LDH/NSE release in H/R injury models)

  • Perform electrophysiological recordings (e.g., BKCa currents using whole-cell patch-clamp technique)

• Controls and validation:

  • Y722F mutant serves as a non-phosphorylatable control

  • Compare wild-type vs. mutant responses to inducers

  • Validate antibody specificity using appropriate negative controls

This comprehensive approach allows researchers to directly link ROCK2 Y722 phosphorylation to specific cellular functions and signaling pathways.