ETC2 Antibody

What is ECT2 and what cellular functions does it regulate?

ECT2 functions as a guanine nucleotide exchange factor (GEF) that catalyzes the exchange of GDP for GTP. It promotes guanine nucleotide exchange on multiple members of the Rho family of small GTPases, including RHOA, RHOC, RAC1, and CDC42. ECT2 is required for signal transduction pathways involved in the regulation of cytokinesis and serves as a component of the centralspindlin complex. This complex mediates microtubule-dependent and Rho-mediated signaling essential for myosin contractile ring formation during cell cycle cytokinesis. Additionally, ECT2 regulates the translocation of RHOA from the central spindle to the equatorial region, plays a role in mitotic spindle assembly by regulating CDC42 activation in metaphase, and participates in epithelial cell polarity through tight junction formation. ECT2 has also been implicated in neurite outgrowth regulation and cancer cell proliferation and invasion through its stimulation of RAC1 activity via association with the oncogenic PARD6A-PRKCI complex .

What types of ECT2 antibodies are available for research applications?

The primary types available include monoclonal antibodies like the mouse monoclonal anti-ECT2 [OTI2D9] which recognizes specific epitopes within human ECT2 (amino acids 150-450). These antibodies are validated for multiple applications including Western blotting (WB) and immunohistochemistry on paraffin-embedded tissues (IHC-P), with confirmed reactivity against human samples. The specificity of these antibodies stems from their development against recombinant fragment proteins encompassing specific regions of the ECT2 sequence . When selecting an antibody, researchers should consider the experimental application, species compatibility, and the specific domain of ECT2 being targeted, as these factors significantly impact experimental outcomes and data interpretation.

How does ECT2 contribute to cancer progression?

ECT2 plays multiple roles in cancer progression through its regulation of small GTPases. It stimulates the activity of RAC1 through association with the oncogenic PARD6A-PRKCI complex in cancer cells, thereby coordinately driving tumor cell proliferation and invasion. Paradoxically, ECT2 also stimulates genotoxic stress-induced RHOB activity in breast cancer cells, which can lead to their cell death . This dual functionality makes ECT2 a complex target for cancer research, requiring careful experimental design to elucidate its context-dependent roles. When investigating ECT2 in cancer models, researchers should consider simultaneously examining downstream effectors like RAC1 and RHOB to fully characterize the signaling networks being affected.

What are the optimal conditions for using ECT2 antibodies in Western blotting?

For optimal Western blotting with ECT2 antibodies, begin with proper sample preparation: lyse cells in a buffer containing protease inhibitors to prevent degradation of ECT2 (MW: ~100 kDa). Use 20-40 μg of total protein per lane and separate on an 8-10% SDS-PAGE gel to achieve proper resolution in the 100 kDa range. Transfer to PVDF membranes (rather than nitrocellulose) for better protein retention. Block with 5% non-fat milk or BSA in TBST for 1 hour at room temperature. Incubate with anti-ECT2 primary antibody (such as clone OTI2D9) at a 1:500-1:1000 dilution overnight at 4°C . After washing, use an appropriate HRP-conjugated secondary antibody at 1:5000 dilution. For enhanced specificity, include positive controls (cell lines known to express ECT2) and negative controls (cell lines with ECT2 knockdown). The expected band should appear at approximately 100-104 kDa, with potential variations due to post-translational modifications.

How can I optimize immunohistochemistry protocols for ECT2 detection in tissue samples?

For effective immunohistochemical detection of ECT2 in paraffin-embedded tissues, start with appropriate antigen retrieval methods. Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes is generally effective for ECT2 antibodies. After blocking endogenous peroxidase activity with 0.3% hydrogen peroxide and non-specific binding with 5% normal serum, apply the ECT2 primary antibody (such as clone OTI2D9) at an optimized dilution (typically 1:100 to 1:200) and incubate overnight at 4°C . Use a biotin-free detection system to minimize background signal. Include proper controls: positive control tissues known to express ECT2 (epithelial tissues or specific cancer tissues) and negative controls by omitting primary antibody. For multiplex immunohistochemistry protocols involving ECT2, sequential staining with appropriate antibody stripping between rounds is recommended to prevent cross-reactivity. Counterstain with hematoxylin for nuclear visualization, keeping staining time brief to avoid masking specific nuclear ECT2 signal.

What cell types and tissues are most appropriate as positive controls for ECT2 antibody validation?

The most appropriate positive controls for ECT2 antibody validation include epithelial cell lines (such as HeLa, MCF-7, or A549) and tissues with high proliferative capacity (such as intestinal epithelium or germinal centers in lymphoid tissue). ECT2 expression is particularly prominent during mitosis due to its critical role in cytokinesis, making highly proliferative cancer cell lines excellent positive controls . For tissue controls, epithelial tissues with high turnover rates are recommended. When validating a new ECT2 antibody, a comparison with previously validated antibodies through parallel staining of the same control samples is advisable. Additionally, using cells or tissues with genetic manipulation of ECT2 (overexpression or knockdown) provides the most stringent validation. For immunohistochemistry applications, normal intestinal epithelium typically shows moderate to strong nuclear and cytoplasmic ECT2 staining, while connective tissue stroma should show minimal background staining.

What are common issues when using ECT2 antibodies in immunoprecipitation experiments?

When performing immunoprecipitation (IP) with ECT2 antibodies, researchers commonly encounter several challenges. First, the efficiency of ECT2 precipitation may be compromised if the antibody's epitope is masked by ECT2's conformational changes during different cell cycle phases or by post-translational modifications. To address this, consider using multiple antibodies targeting different ECT2 epitopes. Second, ECT2's interactions with binding partners like centralspindlin components or GTPases may interfere with antibody recognition. In such cases, adjust lysis conditions by testing different buffers (RIPA vs. NP-40) and salt concentrations (150-500 mM NaCl). Third, cross-reactivity with structurally similar GEF proteins can contaminate IP results. Validate specificity through appropriate controls, including ECT2-depleted lysates . For co-immunoprecipitation of ECT2 with its binding partners, gentler lysis conditions (0.5% NP-40 with 150 mM NaCl) better preserve protein-protein interactions. Finally, when analyzing IP results, remember that ECT2 undergoes cell cycle-dependent phosphorylation, which may cause molecular weight shifts on SDS-PAGE.

How can I apply ECT2 antibodies in studying cytokinesis and cell division mechanisms?

To effectively study cytokinesis and cell division using ECT2 antibodies, combine immunofluorescence microscopy with cell synchronization techniques. For optimal visualization of ECT2's dynamic localization during mitosis, synchronize cells at the G2/M boundary using RO-3306 (CDK1 inhibitor) followed by release, or at metaphase using nocodazole followed by release. Fix cells at different time points post-release using 4% paraformaldehyde and permeabilize with 0.2% Triton X-100. Immunostain with anti-ECT2 antibody along with markers for mitotic structures (α-tubulin for spindle microtubules, Aurora B for the central spindle, and RhoA for the contractile ring) . For high-resolution imaging, apply structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy to resolve ECT2's precise localization at the central spindle and equatorial cortex during anaphase and telophase. To directly link ECT2 function to cytokinesis, combine immunofluorescence with live-cell imaging of cells expressing fluorescently-tagged ECT2 variants (wild-type vs. GEF-dead mutants). For quantitative analysis, measure the correlation between ECT2 intensity at the central spindle/midbody and successful completion of cytokinesis.

How can ChIP-seq protocols be optimized when studying ECT2-associated regulatory factors?

When optimizing ChIP-seq protocols for studying ECT2-associated regulatory factors, several technical considerations are crucial. Although ECT2 itself is not a transcription factor, its interaction with or regulation by transcription factors can be investigated using ChIP-seq with appropriate antibodies against these factors. For instance, when studying how ETS-family transcription factors might regulate ECT2 expression, select antibodies with demonstrated efficacy in ChIP applications . Pre-test multiple antibodies through small-scale ChIP-qPCR experiments before proceeding to sequencing. For fixation, use 1% formaldehyde for 10 minutes at room temperature, as longer fixation can impair epitope accessibility. For sonication, optimize conditions to generate DNA fragments between 200-500 bp, which is ideal for high-resolution mapping of binding sites. Include appropriate controls, such as input DNA and IgG control immunoprecipitations. For data analysis, use peak calling algorithms suitable for transcription factor ChIP-seq (such as MACS2) and validate findings through motif analysis and comparison with published datasets. Integration with RNA-seq data can provide functional context for identified regulatory interactions affecting ECT2 expression.

How should researchers interpret ECT2 expression levels across different cancer types?

When interpreting ECT2 expression across cancer types, researchers should implement a multi-layered analytical approach. First, establish baseline ECT2 expression in corresponding normal tissues using both antibody-based methods (Western blot, IHC) and mRNA analysis (qRT-PCR). For immunohistochemical analysis, use a standardized scoring system (e.g., H-score or Allred score) to quantify both staining intensity and percentage of positive cells . Second, correlate ECT2 expression with clinicopathological parameters including tumor grade, stage, and patient survival data. Third, determine ECT2's subcellular localization, as mislocalization from its normal cytokinesis-related pattern to aberrant nuclear or cytoplasmic accumulation may indicate pathological roles beyond cell division. Fourth, analyze ECT2 in the context of its signaling network by simultaneously assessing levels of downstream effectors (RHOA, RAC1, CDC42) and upstream regulators. Finally, integrate ECT2 protein data with genomic alterations such as gene amplification, which may explain overexpression cases. For comprehensive cancer studies, perform tissue microarray analysis across multiple tumor types to identify cancer-specific patterns of ECT2 dysregulation.

What controls should be included when validating ECT2 antibody specificity for reliable research results?

Comprehensive validation of ECT2 antibody specificity requires multiple complementary controls. First, include genetic controls: compare staining between wild-type cells and those with CRISPR/Cas9-mediated ECT2 knockout or siRNA knockdown—signal should be substantially reduced or eliminated in the latter . Second, perform peptide competition assays where pre-incubating the antibody with the immunizing peptide should abolish specific staining. Third, validate across multiple applications: an antibody showing the expected ~100 kDa band in Western blot, correct subcellular localization in immunofluorescence (central spindle and midbody during cytokinesis), and appropriate tissue distribution in IHC provides stronger evidence of specificity than validation in a single application. Fourth, compare results from multiple antibodies targeting different ECT2 epitopes—concordant results increase confidence in specificity. Fifth, include isotype control antibodies matched to your ECT2 antibody to distinguish non-specific binding. Finally, perform cross-species validation if the antibody is reported to recognize ECT2 from multiple species, confirming consistent molecular weight shifts and expression patterns that align with evolutionary conservation of the protein.

How can researchers differentiate between ECT2's cytokinesis-related functions and its roles in cancer progression?

Differentiating between ECT2's cytokinesis-related functions and its cancer-specific roles requires strategic experimental approaches. First, employ temporal manipulation of ECT2 using inducible knockdown or overexpression systems, allowing distinction between immediate effects on cell division versus long-term effects on transformation. Second, utilize structure-function analysis with domain-specific mutants: compare GEF-dead mutants (affecting both cytokinesis and oncogenic functions) with mutations in cancer-specific interaction domains (e.g., those mediating PARD6A-PRKCI complex binding) . Third, conduct correlation analyses between ECT2 expression/localization and proliferation markers (Ki-67, phospho-histone H3) versus invasion markers (MMPs, EMT transcription factors) in patient samples. Fourth, perform rescue experiments in ECT2-depleted cells using wild-type versus mutant ECT2 to identify which domains are essential for specific cellular phenotypes. Fifth, use synchronized cell populations to distinguish cell cycle-dependent functions from cell cycle-independent oncogenic activities. Finally, investigate ECT2's interactome in normal versus cancer cells using proximity labeling methods (BioID, APEX) combined with mass spectrometry to identify cancer-specific protein interactions. This multi-faceted approach enables researchers to clearly delineate ECT2's dual roles in normal cell division and pathological cancer progression.

What are the methodological approaches for studying ECT2's role in regulating epithelial cell polarity?

To investigate ECT2's role in epithelial cell polarity, researchers should employ a multi-faceted methodological approach. Begin with 3D cell culture systems using Matrigel or collagen matrices, which better recapitulate epithelial architecture than traditional 2D cultures. In these systems, manipulate ECT2 expression through inducible shRNA or CRISPR/Cas9 and assess polarity markers including apical (ezrin, aPKC), basolateral (Na+/K+-ATPase, E-cadherin), and tight junction (ZO-1, occludin) proteins . For mechanistic insights, perform co-immunoprecipitation studies to analyze ECT2's interactions with polarity complex proteins (PARD3, PARD6, aPKC). Use super-resolution microscopy (STORM, STED) to visualize the precise localization of ECT2 relative to junctional complexes during polarization. For functional assays, measure transepithelial electrical resistance (TEER) to assess junction integrity and employ calcium switch assays to study dynamic junction assembly and disassembly. Additionally, utilize FRET biosensors to monitor the spatiotemporal activation of ECT2's downstream GTPases (CDC42, RAC1) during polarization events. Finally, validate findings in organoid models derived from primary tissues, which provide a physiologically relevant context for studying epithelial organization under conditions where ECT2 is manipulated.

How can researchers integrate ECT2 antibody staining with live-cell imaging techniques for studying dynamic cell processes?

Integrating ECT2 antibody staining with live-cell imaging requires sophisticated technical approaches that bridge fixed and dynamic cellular analyses. Start by establishing stable cell lines expressing fluorescently-tagged ECT2 (e.g., ECT2-GFP) at near-endogenous levels using CRISPR knock-in strategies rather than overexpression systems. Validate these reporter lines by fixed-cell immunofluorescence, confirming that the tagged ECT2 colocalizes with antibody-detected endogenous ECT2 and exhibits expected localization patterns during cell division . For correlative live-cell imaging and immunofluorescence (CLEM), use gridded coverslips or dishes with fiducial markers to track specific cells through both imaging modalities. Perform live imaging of fluorescently-tagged ECT2 dynamics, capturing key events (e.g., central spindle accumulation, cortical recruitment), then immediately fix cells for antibody staining against additional markers impossible to visualize in live cells. For pulse-chase analyses of ECT2 turnover, combine live imaging with techniques like fluorescence recovery after photobleaching (FRAP) or photoactivatable fluorescent proteins. When studying ECT2's role in rapid GTPase activation, pair ECT2-RFP with FRET-based biosensors for RhoA, CDC42, or RAC1 activation. Finally, for long-term tracking of ECT2 dynamics in development or 3D culture systems, use selective plane illumination microscopy (SPIM) with minimal phototoxicity, allowing correlation between ECT2 localization patterns and resulting morphological changes.

What approaches should be used to investigate contradictory findings regarding ECT2's role in specific cellular processes?

When investigating contradictory findings regarding ECT2's cellular roles, researchers should implement a systematic reconciliation strategy. First, perform careful methodological cross-comparison between conflicting studies, examining differences in cell types, ECT2 manipulation approaches (siRNA, CRISPR, overexpression), and experimental conditions that might explain discrepancies . Second, conduct simultaneous phenotypic analyses using multiple, complementary readouts rather than relying on single assays. For instance, when studying ECT2's effect on cell migration, combine scratch assays, single-cell tracking, transwell migration, and 3D invasion assays. Third, employ dose-dependent and temporal analyses, as ECT2 may exhibit biphasic effects depending on expression level or timing of manipulation. Fourth, examine cell type specificity by testing identical experimental setups across multiple cell lines, primary cells, and in vivo models. Fifth, use domain-specific mutants and chimeric constructs to determine which ECT2 domains are responsible for potentially opposing functions. Sixth, investigate context dependency by manipulating microenvironmental factors (ECM composition, stiffness, growth factors) that might influence ECT2's functional output. Finally, perform unbiased multi-omics analyses (proteomics, transcriptomics) on samples with ECT2 manipulation to identify cell-type specific downstream pathways that might explain contradictory phenotypic outcomes in different experimental systems.

What methodologies are emerging for studying ECT2's interactions with the centralspindlin complex during cytokinesis?

Cutting-edge methodologies for studying ECT2-centralspindlin interactions during cytokinesis combine advanced imaging with innovative biochemical approaches. Super-resolution microscopy techniques (3D-SIM, PALM, STORM) now enable visualization of nanoscale spatial relationships between ECT2 and centralspindlin components (MKLP1, MgcRacGAP) at the midbody with <50 nm resolution. For temporal dynamics, lattice light-sheet microscopy offers unprecedented spatiotemporal resolution for tracking ECT2 recruitment during anaphase progression . At the molecular level, researchers are applying proximity labeling methods (BioID, TurboID, APEX) to identify the complete interactome of ECT2 specifically during cytokinesis. These approaches can be combined with synchronization strategies and rapid induction systems (e.g., auxin-inducible degron) to achieve precise temporal control. For structural insights, cryo-electron microscopy is being applied to resolve ECT2-centralspindlin complex structures, while hydrogen-deuterium exchange mass spectrometry (HDX-MS) helps map conformational changes upon complex formation. Functionally, optogenetic tools for spatiotemporally controlled activation or inhibition of ECT2 at the central spindle allow direct testing of causality in centralspindlin-mediated cytokinesis signaling. Finally, in vitro reconstitution assays using purified components and artificial membrane systems enable mechanistic dissection of how ECT2-centralspindlin interactions promote RhoA activation and contractile ring assembly in a controlled environment.

How can multi-omics approaches help elucidate ECT2's broader role in cellular signaling networks?

Multi-omics approaches offer powerful strategies for contextualizing ECT2 within broader cellular signaling networks. Integrate phosphoproteomics with ECT2 manipulation (knockdown/overexpression) to map how ECT2 impacts phosphorylation cascades, particularly those involving its downstream GTPases and their effectors. This should be performed across multiple timepoints and cell cycle stages to capture dynamic signaling events . Combine this with interactome analysis using BioID or immunoprecipitation-mass spectrometry to identify cell cycle-specific and cell type-specific ECT2 binding partners. Chromatin immunoprecipitation sequencing (ChIP-seq) of transcription factors regulated downstream of ECT2-activated GTPases can reveal indirect transcriptional programs governed by ECT2 activity. Complement these approaches with RNA-seq to determine gene expression changes following ECT2 perturbation, and metabolomics to assess whether ECT2's roles extend to metabolic regulation through its GTPase targets. For spatial context, employ imaging mass cytometry to map ECT2 expression patterns alongside dozens of signaling proteins in tissue samples. Finally, integrate these multi-omics datasets using computational approaches like weighted gene co-expression network analysis (WGCNA) or partial least squares regression to build predictive models of ECT2-dependent signaling networks. This integrative approach can reveal unexpected connections between ECT2 and other cellular processes beyond its established roles in cytokinesis and cell polarity.