DIAPH2 Antibody

What is DIAPH2 and what cellular functions does it regulate?

DIAPH2, also known as DRF2 (diaphanous-related formin 2), is a 1101 amino acid cytoplasmic protein with a molecular weight of approximately 126-130 kDa that belongs to the Diaphenous family of actin binding proteins . DIAPH2 plays multiple roles in cellular processes, with evidence supporting its involvement in:

• Regulation of spindle microtubule dynamics during M-phase cell division, where it controls chromosome alignment and movement velocity

• Endosome dynamics regulation, particularly through interactions with the actin cytoskeleton

• A novel signal transduction pathway in which specific isoforms (particularly isoform 3) interact with RHOD and CSK to regulate early endosome motility

• Potential involvement in oogenesis, as disruption of DIAPH2 has been correlated with premature ovarian failure in humans

• Possible role in inner ear development, though knockout mouse models have not demonstrated clear hearing impairment

The protein has multiple functional domains, with the FH2 domain being crucial for microtubule dynamics regulation, though research suggests there may be additional microtubule-binding regions within the protein .

What applications are DIAPH2 antibodies validated for?

DIAPH2 antibodies have been validated for several research applications, though the extent of validation varies by manufacturer and antibody clone. The primary validated applications include:

• Western Blotting (WB): Multiple antibodies have demonstrated successful detection of DIAPH2 protein in human cell lines, with observed bands at approximately 130 kDa . Specific validation has been documented in cell lysates from HeLa, HCT-116, and 293T cell lines .

• Immunohistochemistry (IHC): Antibodies have been successfully used for detection of DIAPH2 in formalin-fixed paraffin-embedded tissue sections, particularly in mouse cochlear samples .

• Immunofluorescence (IF): DIAPH2 antibodies have been used to visualize the protein's cellular localization, revealing diffuse cytosolic distribution during interphase and spindle microtubule localization during metaphase .

When selecting an antibody for your research, verify that the specific clone has been validated for your application of interest, as performance can vary significantly between applications even for the same antibody .

What is the expected molecular weight and band pattern for DIAPH2 in Western blot analysis?

When performing Western blot analysis for DIAPH2, researchers should expect to observe:

• A primary band at approximately 130 kDa, which has been consistently reported in validated Western blots

• The predicted molecular weight based on amino acid sequence is 126 kDa, but the observed weight is typically around 130 kDa, likely due to post-translational modifications

It's important to note that multiple isoforms of DIAPH2 have been described in the literature:

• The canonical form consists of 1101 amino acids

• An isoform called hDia2C (1097 aa) lacks amino acids 45-55 but has 7 additional amino acids inserted after position 149; this form specifically binds RhoD and regulates early endosome movement

• A 1106 aa form has been reported that diverges after amino acid 1080

These isoform variations may result in subtle band size differences or multiple bands depending on the epitope recognized by the antibody and the sample type being analyzed. The Western blot data from Abcam's antibody (ab181165) shows a single band at approximately 130 kDa in HeLa, HCT-116, and 293T cell lysates .

Which species are DIAPH2 antibodies reactive with?

Based on the available search results, DIAPH2 antibodies show these species reactivities:

For cross-species applications, it's worth noting that within the immunogen region used for the R&D Systems antibody, human DIAPH2 shares 89% and 90% amino acid identity with mouse and rat DIAPH2, respectively . This suggests potential cross-reactivity, though explicit validation would be required before use in these species.

When considering antibodies for non-human applications, verify the sequence homology between species in the region recognized by the antibody. Even with high sequence homology, cross-reactivity should be experimentally validated before proceeding with critical experiments .

How should I optimize Western blot conditions for DIAPH2 detection?

Based on the documented protocols in the literature, the following optimization strategies are recommended for DIAPH2 Western blotting:

• Sample Preparation:

  • Use reducing conditions, as successful detection has been documented under these conditions

  • For human samples, HEK293, HeLa, and HCT-116 cell lysates have shown reliable DIAPH2 expression

  • The standard sample loading amount used in validated studies is approximately 10 μg of total protein lysate

• Blocking and Buffer Conditions:

  • The R&D Systems protocol specifies using "Immunoblot Buffer Group 2," suggesting specific buffer requirements may improve results

  • Standard blocking with 5% non-fat dry milk or BSA in TBS-T is a reasonable starting point

• Antibody Concentrations:

  • Primary antibody: R&D Systems recommends 2 μg/mL of their mouse monoclonal antibody

  • Abcam recommends a 1:1000 dilution of their rabbit monoclonal antibody

  • Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG at 1:1000 dilution has proven effective

• Detection Method:

  • Enhanced chemiluminescence (ECL) is suitable for detecting the 130 kDa DIAPH2 band

  • Exposure times may need optimization based on expression levels in your specific samples

• Controls:

  • Positive controls: HEK293, HeLa, or HCT-116 cell lysates are appropriate positive controls

  • Testis tissue lysate has also been validated for DIAPH2 detection

For troubleshooting, if non-specific bands appear, increasing the stringency of washing steps and optimizing antibody concentrations may improve specificity. If no signal is detected, verify protein transfer efficiency and consider longer exposure times or more sensitive detection methods.

What are the optimal fixation and staining protocols for DIAPH2 immunocytochemistry?

Based on the protocols described in the literature, the following recommendations can be made for DIAPH2 immunocytochemistry:

• Cell Fixation:

  • For cultured cells (e.g., HEK293), fixation with 4% paraformaldehyde for 20 minutes at room temperature has been successful

  • Post-fixation, perform a brief permeabilization step with 0.1% Triton X-100 in PBS for 5 minutes

• Blocking:

  • Block with a solution containing 10% fetal bovine serum, 2% bovine serum albumin, 0.1% Triton-X100 in PBS for 1 hour at room temperature

• Antibody Incubation:

  • Primary antibody: Dilutions will depend on the specific antibody used. For the Santa Cruz Diaph2 antibody (sc-10892), a 1:50 dilution in blocking buffer has proven effective

  • Incubate with primary antibody for approximately 1 hour at room temperature or overnight at 4°C

  • Secondary antibody: Fluorophore-conjugated secondary antibodies (such as Dylight 549 anti-mouse or Alexa Fluor 633 anti-rabbit) at 1:200 dilution for 1 hour at room temperature

• Washing and Counterstaining:

  • Wash 3 times with high salt buffer (500 mM NaCl, 20 mM Na-phosphate buffer pH 7.4), followed by low salt buffer (150 mM NaCl, 10 mM phosphate buffer pH 7.4)

  • For nuclear counterstaining, DAPI can be used (5-minute incubation)

  • For F-actin co-staining, ActinGreen 488 or similar reagents are appropriate

• Imaging Parameters:

  • Confocal microscopy with a 63x oil-immersion objective (N.A. 1.40) has been successfully used to visualize DIAPH2 localization

  • For optimal resolution, set confocal aperture to 1 airy unit

  • Maintain identical gain, offset, exposure, and laser-power settings between experimental conditions for valid comparisons

For tissue immunohistochemistry, a modified protocol has been documented:

• Use 8 μm thick formalin-fixed paraffin-embedded sections

• Perform heat-induced epitope retrieval prior to antibody incubation

• Incubate with primary antibody (1:50) for 1 hour, followed by biotinylated secondary antibody (1:100) for 16 minutes

How can I verify the specificity of my DIAPH2 antibody?

Verifying antibody specificity is crucial for ensuring reliable research findings. For DIAPH2 antibodies, consider implementing these validation approaches:

• Western Blot Band Size Verification:

  • Confirm that the detected band appears at the expected molecular weight of approximately 130 kDa

  • Compare to reported band patterns in literature and manufacturer datasheets

• Positive and Negative Controls:

  • Positive controls: Use cell lines with documented DIAPH2 expression like HEK293, HeLa, or HCT-116

  • Negative/knockdown controls: If available, test the antibody on DIAPH2 knockdown/knockout cells generated using siRNA or CRISPR/Cas9 technology

• Recombinant Protein Controls:

  • Test antibody recognition of recombinant DIAPH2 protein. The R&D Systems antibody was raised against E. coli-derived recombinant human DIAPH2 (Val314-Gln540)

  • Compare detection of wild-type versus mutant DIAPH2 (such as the c.868A>G variant) to assess epitope specificity

• Cross-reactivity Assessment:

  • Test for cross-reactivity with other diaphanous family members (DIAPH1, DIAPH3) which share structural similarity

  • This is particularly important as these family members have been associated with similar cellular functions

• Immunofluorescence Pattern Verification:

  • Confirm that the subcellular localization pattern matches expected distribution: diffuse cytosolic in interphase cells and spindle microtubule localization during metaphase

  • Co-localization studies with known markers can provide further validation

• Blocking Peptide Competition:

  • If available, preincubate the antibody with a blocking peptide corresponding to the immunogen and verify signal disappearance in both Western blot and immunostaining

Implementing multiple validation approaches provides stronger evidence for antibody specificity than relying on a single method. Document these validation steps thoroughly in your methods section when publishing results.

How can I use DIAPH2 antibodies to investigate its role in microtubule dynamics?

Based on the research findings, DIAPH2 plays a significant role in microtubule dynamics, particularly during spindle formation in mitosis . Here are methodological approaches to investigate this function:

• Live-Cell Imaging with Fluorescently Tagged Proteins:

  • Transfect cells with fluorescently tagged DIAPH2 (GFP-DIAPH2) and fluorescently tagged tubulin (mCherry-tubulin)

  • Use time-lapse confocal microscopy to track co-localization during different cell cycle phases

  • Measure parameters such as the velocity of chromosome movement, which has been shown to decrease with Diaph2 down-regulation

• Microtubule Stability Assays:

  • Compare the stability of spindle microtubules between control and DIAPH2-depleted cells using cold-treatment assays (cold-stable microtubules represent the stable subset)

  • Research has shown that Diaph2-depletion increases the concentration of stable spindle microtubules

  • Quantify relative fluorescence intensity of microtubules before and after cold treatment

• In Vitro Microtubule Polymerization Assays:

  • Use purified tubulin and recombinant DIAPH2 proteins to assess direct effects on microtubule polymerization rates

  • Compare full-length DIAPH2 with domain-specific constructs to identify functional regions

  • Full-length DIAPH2 has been shown to mediate a 10-fold increase in MT-polymerization compared to just the FH2-domain

• Domain-Specific Function Analysis:

  • Generate and express DIAPH2 constructs with specific domain deletions (e.g., ΔFH2) to distinguish the roles of different protein regions

  • Research has suggested that regions outside the canonical FH2 domain may also affect microtubule dynamics

  • Functional readouts should include: mitotic progression, spindle morphology, and microtubule polymerization rates

• RhoA-Independence Testing:

  • Since DIAPH2's effect on microtubules appears to be independent of Cdc42/RhoA activity (which normally regulates its actin-nucleating function), design experiments with constitutively active or dominant negative RhoA/Cdc42 mutants

  • Compare microtubule effects in these backgrounds to confirm pathway independence

The experimental data should be quantified rigorously, measuring parameters such as:

• Microtubule polymerization rates (nm/sec)

• Spindle microtubule density

• Chromosome alignment efficiency (% aligned chromosomes)

• Mitotic progression timing (minutes from prophase to anaphase)

What experimental approaches can reveal DIAPH2's role in hearing and inner ear development?

The search results indicate that DIAPH2 may play a role in the inner ear, although initial knockout mouse studies did not show clear hearing impairment . To further investigate this potential function, consider these methodological approaches:

• Temporal and Spatial Expression Analysis:

  • Perform detailed immunohistochemistry of mouse inner ear development at multiple timepoints (embryonic to adult stages)

  • Research has shown that Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of sensory outer hair cells

  • Use cochlear whole-mount preparations for better visualization of hair cell stereocilia

• More Sensitive Auditory Testing:

  • While initial ABR (Auditory Brainstem Response) measurements at 4, 8, and 14 weeks in knockout mice showed no obvious impairment, more sensitive methods might reveal subtle defects

  • Implement Distortion Product Otoacoustic Emissions (DPOAE) testing, which specifically assesses outer hair cell function

  • Consider testing at higher frequencies or under stress conditions (noise exposure, aging)

• Structural Analysis of Stereocilia:

  • Since Diaph2 localizes to stereocilia and is involved in actin dynamics, perform detailed morphological analysis using:

    • Scanning electron microscopy (SEM) to assess stereocilia bundle architecture

    • Measurement of stereocilia length, width, and organization in knockout vs. wild-type mice

    • Quantification of F-actin content and organization using fluorescent phalloidin staining

• Conditional and Cell-Type Specific Knockouts:

  • Generate conditional knockouts (e.g., using hair-cell specific Cre lines) to address potential compensatory mechanisms in the global knockout

  • Consider double knockouts of multiple diaphanous family members, as functional redundancy may mask phenotypes

• Human Genetic Studies:

  • Follow up on the identified missense variant (c.868A>G) that segregated with nonsyndromic X-linked hearing loss in an Italian family

  • Implement larger scale sequencing studies of DIAPH2 in patients with unexplained hearing loss

  • Develop functional assays for testing the effects of patient-derived variants on protein function

• In Vitro Functional Studies:

  • Test whether the c.868A>G mutation or other variants affect RhoA-dependent activation of DIAPH2

  • Assess the impact on actin polymerization in hair cell models

  • Use techniques like FRAP (Fluorescence Recovery After Photobleaching) to evaluate actin dynamics in cells expressing wild-type vs. mutant DIAPH2

A combined approach using these methodologies would provide more comprehensive insights into DIAPH2's potential role in hearing.

How can I design experiments to investigate DIAPH2's interactions with RhoA and its role in signal transduction?

DIAPH2 functions in signal transduction pathways involving Rho GTPases like RhoA and RhoD . To investigate these interactions and their functional consequences, consider these methodological approaches:

• Co-Immunoprecipitation (Co-IP) Studies:

  • Use anti-DIAPH2 antibodies to immunoprecipitate the protein complex from cells

  • Probe for co-precipitated RhoA/RhoD using specific antibodies

  • Compare binding efficiency under different cellular conditions:

    • Serum stimulation vs. starvation

    • GTPase-activating vs. inhibitory conditions

  • Include appropriate controls (IgG control, input lysate)

• In Vitro Binding Assays with Purified Components:

  • Express and purify recombinant DIAPH2 (full-length and domain-specific constructs)

  • Perform pull-down assays with GST-tagged constitutively active (G14V) or dominant negative RhoA/RhoD

  • Quantify binding affinity using surface plasmon resonance or microscale thermophoresis

• Cellular Localization Studies:

  • Co-transfect cells with tagged DIAPH2 and constitutively active RhoA (RhoA-G14V)

  • Use confocal microscopy to analyze co-localization patterns

  • Quantify changes in DIAPH2 localization in response to RhoA activation or inhibition

  • Document any changes in membrane protrusion formation (which has been shown to differ between wild-type and mutant DIAPH2 expression)

• Functional Readouts of DIAPH2-RhoA Interaction:

  • Assess actin polymerization using pyrene-actin assays with purified components

  • Compare the effects of wild-type DIAPH2 versus mutant forms (such as the c.868A>G variant)

  • Quantify cellular phenotypes like membrane protrusion length, which showed significant differences between wild-type and mutant DIAPH2 expressing cells

• Domain Mapping of Critical Interaction Regions:

  • Generate truncation mutants of DIAPH2 to identify which domains are essential for RhoA binding

  • Special attention should be paid to the DID (Diaphanous Inhibitory Domain) which typically interacts with Rho GTPases in formin proteins

  • Monitor effects of domain mutations on both binding and downstream functions

• Signal Transduction Pathway Analysis:

  • Investigate the sequential activation of DIAPH2 and CSK by RhoD as described for endosome regulation

  • Use phospho-specific antibodies to monitor activation states of pathway components

  • Implement proximity ligation assays (PLA) to visualize protein-protein interactions in situ

These experimental approaches should provide comprehensive insights into DIAPH2's interactions with Rho GTPases and their functional consequences in cellular signaling pathways.