SHROOM2 Antibody, FITC conjugated

What cellular functions does SHROOM2 regulate that make it a valuable research target?

SHROOM2 serves as a key mediator of the RhoA-ROCK pathway, regulating cell motility and actin cytoskeleton organization. Beyond this pathway, SHROOM2 plays a critical role in suppressing epithelial-to-mesenchymal transition (EMT) and tumor metastasis through ROCK-independent mechanisms . The protein contains specific domains - SHROOM-domain 1 (SD1) and SHROOM-domain 2 (SD2) - that bind F-actin and ROCK respectively, enabling it to recruit ROCK to the cytoskeleton and induce phosphorylation of non-muscle myosin II for cellular morphology remodeling . These diverse functions make SHROOM2 particularly valuable for research in developmental biology, cancer biology, and cellular migration studies.

What are the recommended applications for FITC-conjugated SHROOM2 antibodies?

FITC-conjugated SHROOM2 antibodies are primarily optimized for immunofluorescence (IF) applications, allowing direct visualization of SHROOM2 protein localization without requiring secondary antibody incubation. The FITC conjugation enables efficient detection in fluorescence microscopy with excitation at approximately 495 nm and emission at around 519 nm. These antibodies can be effectively used for immunofluorescence staining in fixed cells, as demonstrated in MCF-7 cells where subcellular distribution patterns can be clearly observed when counter-stained with DAPI . Additionally, these conjugated antibodies may be suitable for flow cytometry applications to quantify SHROOM2 expression in cell populations, though specific validation for this application may be required.

What is the recommended protocol for immunofluorescence staining using FITC-conjugated SHROOM2 antibody?

For optimal immunofluorescence results with FITC-conjugated SHROOM2 antibody, researchers should follow this validated protocol: First, fix cells in 4% formaldehyde for 15 minutes at room temperature. After washing with PBS, permeabilize the cells using 0.2% Triton X-100 for 10 minutes. Block non-specific binding by incubating cells with 10% normal goat serum for 1 hour . Dilute the FITC-conjugated SHROOM2 antibody at 1:50-1:200, with 1:66 being an optimal dilution for many cell types, and incubate overnight at 4°C in a humidified chamber . After washing thoroughly with PBS, counterstain with DAPI to visualize nuclei. Mount slides with anti-fade mounting medium and examine using a fluorescence microscope with appropriate filter sets for FITC detection. This protocol has been validated for detecting endogenous SHROOM2 in various cell lines including MCF-7 cells.

How can researchers effectively distinguish between ROCK-dependent and ROCK-independent functions of SHROOM2 in experimental designs?

To differentiate between ROCK-dependent and ROCK-independent functions of SHROOM2, researchers should implement a multi-faceted experimental approach. Start by establishing SHROOM2 knockdown cell lines using specific shRNAs (three validated sequences are available: 5′-GATGAGATCGTCGGCATCAAT-3′, 5′-GAGCGCATCGTCTTTGACATT-3′, and 5′-CCACCAATTCTACCTACTACA-3′) . In parallel experiments, treat both control and SHROOM2-depleted cells with ROCK inhibitor Y-27632. ROCK-dependent functions will show similar phenotypes between SHROOM2 knockdown and Y-27632 treatment, with no additive effects when combined. For instance, both SHROOM2 depletion and Y-27632 treatment similarly reduce stress fiber formation and focal adhesions . Conversely, ROCK-independent functions will show distinct phenotypes between SHROOM2 knockdown and Y-27632 treatment, with potential synergistic effects when combined. This is exemplified by EMT marker expression changes (E-cadherin decrease, N-cadherin increase) that occur with SHROOM2 depletion but not with Y-27632 treatment . Researchers should analyze multiple readouts including cell morphology, protein expression (via Western blot), cytoskeletal organization (via phalloidin staining), and functional assays such as migration and invasion to comprehensively distinguish these pathways.

What are the critical considerations when using FITC-conjugated SHROOM2 antibodies for co-localization studies with ROCK pathway components?

When designing co-localization studies with FITC-conjugated SHROOM2 antibodies and ROCK pathway components, several critical factors must be addressed. First, spectral compatibility is essential - FITC (excitation ~495nm, emission ~519nm) must be paired with fluorophores that have minimal spectral overlap such as Cy5 or Alexa 647 for co-staining ROCK1 or phosphorylated MLC2. Second, researchers must optimize fixation methods, as SHROOM2's membrane localization is particularly sensitive to fixation conditions - 4% formaldehyde fixation preserves membrane structures better than methanol-based protocols . Third, validation controls are necessary: analyze SHROOM2-depleted cells (using verified shRNAs) to confirm antibody specificity, and include Y-27632 ROCK inhibitor treatments to verify functional interactions . Fourth, researchers should quantitatively analyze co-localization using appropriate software and statistical methods rather than relying on qualitative visual assessment alone. Finally, when examining subcellular distribution of ROCK and MLC2 in relation to SHROOM2, membrane fractionation followed by Western blotting can provide biochemical confirmation of imaging results, particularly important since SHROOM2 depletion has been shown to impair membrane recruitment of both ROCK1 and MLC2 .

How can researchers accurately quantify changes in SHROOM2 localization during epithelial-to-mesenchymal transition (EMT) using FITC-conjugated antibodies?

To accurately quantify SHROOM2 localization changes during EMT using FITC-conjugated antibodies, researchers should implement a comprehensive image analysis workflow. Begin with standardized image acquisition parameters, maintaining consistent exposure times, gain settings, and optical sectioning techniques across all experimental conditions. Establish an EMT model system using TGF-β treatment or other EMT inducers, and validate the progression of EMT via expression analysis of canonical markers (E-cadherin, N-cadherin, vimentin) . For quantification, utilize high-content imaging platforms that enable automated analysis of multiple parameters simultaneously: measure integrated FITC intensity at specific subcellular regions (membrane, cytoplasm, cell junctions) using cellular compartment masks defined by co-staining with organelle markers or membrane dyes. Calculate the membrane-to-cytoplasm ratio of SHROOM2 signal as this ratio typically decreases during EMT progression. Additionally, implement advanced analytical methods such as radial intensity profiling from nuclear center to cell periphery to capture redistribution patterns systematically. To ensure reliable quantification, analyze a statistically significant number of cells (minimum 50-100 per condition) across at least three independent experiments, and utilize appropriate statistical tests for comparing distribution patterns between epithelial and mesenchymal states. This approach allows for robust detection of the subcellular redistribution of SHROOM2 that accompanies phenotypic changes during EMT progression.

What is the optimal fixation and permeabilization protocol to preserve SHROOM2 localization at cell-cell junctions and the cytoskeleton?

The optimal fixation and permeabilization protocol for preserving SHROOM2's native localization at cell-cell junctions and cytoskeleton requires careful attention to several parameters. For fixation, use freshly prepared 4% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature - this approach maintains membrane integrity better than methanol-based methods which can disrupt the association of SHROOM2 with the actin cytoskeleton . After fixation, wash cells thoroughly with PBS (3 x 5 minutes) to remove all traces of fixative. For permeabilization, use 0.2% Triton X-100 in PBS for exactly 10 minutes at room temperature - this concentration allows antibody access while preserving delicate cytoskeletal structures . For cells with particularly sensitive junctional complexes, consider a milder alternative permeabilization using 0.1% saponin. Post-permeabilization, a blocking step using 10% normal goat serum is critical to reduce non-specific binding . When studying SHROOM2's association with specific cytoskeletal structures, preservation of F-actin is crucial; therefore, avoid excessive washing steps and mechanical stress during the procedure. For epithelial cells where junctional localization is important, growing cells to appropriate confluence (80-90%) before fixation ensures proper formation of cell-cell contacts where SHROOM2 may concentrate. This protocol has been validated to properly preserve both the membrane/junctional and cytoskeletal pools of SHROOM2, enabling accurate assessment of its subcellular distribution in both normal and pathological contexts.

How should researchers validate the specificity of FITC-conjugated SHROOM2 antibody staining patterns?

Comprehensive validation of FITC-conjugated SHROOM2 antibody specificity requires a multi-pronged approach. First, researchers should perform RNA interference experiments using at least two distinct validated shRNA sequences targeting SHROOM2 (such as 5′-GAGCGCATCGTCTTTGACATT-3′ and 5′-CCACCAATTCTACCTACTACA-3′) . The FITC signal should be significantly reduced in knockdown cells compared to control cells. Second, implement a rescue experiment using shRNA-resistant SHROOM2 constructs generated by introducing silent mutations in the shRNA targeting sequence (e.g., 5′-GAGCGCATTGTTTTCGATATC-3′) and verify restoration of the FITC staining pattern . Third, perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide (such as amino acids 599-625 or 213-405, depending on the antibody) should abolish specific staining. Fourth, compare staining patterns across multiple cell lines with known SHROOM2 expression levels, confirming correlation between staining intensity and protein expression quantified by Western blot. Fifth, perform parallel staining with a second, independent SHROOM2 antibody targeting a different epitope to confirm overlapping localization patterns. Finally, for definitive validation in advanced studies, utilize CRISPR/Cas9 knockout cells as the gold standard negative control. These comprehensive validation steps ensure that the observed FITC staining patterns genuinely reflect SHROOM2 localization rather than non-specific binding or artifacts.

What are the recommended dilutions and incubation conditions for optimal signal-to-noise ratio when using FITC-conjugated SHROOM2 antibodies?

For optimal signal-to-noise ratio when using FITC-conjugated SHROOM2 antibodies, researchers should follow these empirically determined parameters: For immunofluorescence applications, the recommended dilution range is 1:50-1:200 in antibody diluent containing 1-2% BSA and 0.1% Triton X-100 in PBS, with 1:66 being an optimal starting dilution for most cell types . Incubate primary antibody overnight (14-16 hours) at 4°C in a humidified chamber to ensure adequate epitope binding while minimizing background. For ELISA applications, use more dilute concentrations ranging from 1:2000-1:10000 . When optimizing for a new cell type or tissue, perform a titration experiment with multiple dilutions (e.g., 1:25, 1:50, 1:100, 1:200) to determine the optimal concentration that maximizes specific signal while minimizing background. Pre-absorption of the antibody with 5% normal serum from the same species as the experimental samples can further reduce non-specific binding. For washing steps, use PBS with 0.05% Tween-20 and perform at least 3 washes of 5 minutes each after antibody incubation. When imaging, adjust exposure settings to ensure the FITC signal remains within the linear range of detection, avoiding saturation which can mask differences in expression levels. These optimized conditions ensure reproducible detection of SHROOM2 with high specificity and minimal background interference.

How can researchers address potential cross-reactivity issues between SHROOM2 antibodies and other SHROOM family proteins?

Addressing cross-reactivity between SHROOM2 antibodies and other SHROOM family members (SHROOM1, SHROOM3, SHROOM4) requires systematic validation strategies. First, researchers should select antibodies targeting unique regions of SHROOM2 rather than conserved domains - antibodies targeting the central region (amino acids 599-625) or the recombinant 213-405AA region show greater specificity than those targeting the highly conserved SD1 or SD2 domains . Second, perform Western blot analysis across cell lines with differential expression of SHROOM family members to confirm the antibody detects bands of the expected molecular weight for SHROOM2 (approximately 175-180 kDa) without detecting other SHROOM proteins with similar molecular weights (SHROOM3: ~220 kDa, SHROOM4: ~192 kDa). Third, validate specificity using gene silencing approaches with multiple independent SHROOM2-specific shRNAs, which should reduce immunofluorescence signal without affecting other SHROOM family members . Fourth, for definitive cross-reactivity testing, overexpress each SHROOM family member individually in a cell line with low endogenous expression and confirm the antibody only detects SHROOM2. Fifth, utilize mass spectrometry-based validation by immunoprecipitating with the SHROOM2 antibody and confirming the identity of pulled-down proteins. Sixth, when analyzing tissues known to express multiple SHROOM family members, always include appropriate positive and negative controls. These comprehensive approaches ensure that observed signals genuinely represent SHROOM2 rather than cross-reactive detection of other SHROOM family proteins.

How should contradictory data between SHROOM2 protein levels detected by immunofluorescence versus Western blot be reconciled?

When confronted with discrepancies between SHROOM2 levels detected by immunofluorescence (IF) with FITC-conjugated antibodies versus Western blot (WB), researchers should implement a systematic troubleshooting approach. First, consider epitope accessibility differences - IF detects native epitopes while WB detects denatured epitopes, so antibodies recognizing conformational epitopes may perform differently in each application. Verify whether the same antibody clone is being used for both techniques; if using separate antibodies, validate both independently . Second, evaluate fixation effects, as overfixation can mask epitopes in IF while inadequate lysis might affect protein extraction for WB. Third, examine sensitivity thresholds - WB may detect total protein more quantitatively, while IF offers superior spatial resolution but potential heterogeneity in single-cell expression might be averaged out in WB. Fourth, investigate potential post-translational modifications that might affect epitope recognition differently in native versus denatured states. Fifth, validate results with alternative approaches: for WB discrepancies, test different lysis buffers optimized for membrane proteins and confirm loading controls; for IF discrepancies, try alternative fixation protocols and include appropriate positive and negative control cell lines . Sixth, consider using absolute quantification methods such as recombinant protein standards in WB and calibrated fluorescence standards in IF to establish accurate quantitative relationships between the techniques. Finally, use orthogonal methods such as qRT-PCR to determine if discrepancies reflect post-transcriptional regulation. This comprehensive approach enables researchers to determine whether discrepancies represent technical artifacts or biologically meaningful differences in protein expression, modification, or localization.

How can FITC-conjugated SHROOM2 antibodies be effectively used to investigate the protein's role in suppressing tumor metastasis?

To investigate SHROOM2's role in tumor metastasis suppression using FITC-conjugated antibodies, researchers should implement a comprehensive experimental framework spanning in vitro and in vivo models. Begin with comparative immunofluorescence analysis of SHROOM2 localization and expression levels in paired primary tumor and metastatic samples, similar to the approach used in nasopharyngeal carcinoma where metastatic tumors showed significantly lower SHROOM2 levels . Establish stable knockdown cell lines using validated shRNAs (e.g., 5′-GAGCGCATCGTCTTTGACATT-3′) alongside shRNA-resistant rescue constructs to confirm phenotype specificity . Design time-course experiments tracking SHROOM2 redistribution during EMT induction, correlating its localization changes with alterations in E-cadherin, N-cadherin, and other EMT markers. Implement functional migration and invasion assays (transwell, wound healing) comparing control, SHROOM2-depleted, and SHROOM2-overexpressing cells, both with and without ROCK inhibitor Y-27632 to distinguish pathway dependencies . For in vivo studies, utilize both experimental metastasis models (tail vein injection) and spontaneous metastasis models (footpad injection with lymph node metastasis assessment), quantifying metastatic burden through histological and immunohistochemical analysis . Use the FITC-conjugated antibodies for high-resolution intravital imaging to track SHROOM2 dynamics in tumor cells during intravasation and extravasation processes in appropriate animal models. This integrated approach enables comprehensive characterization of SHROOM2's spatial and temporal dynamics during metastatic progression, providing mechanistic insights into its suppressive functions.

What experimental controls should be included when studying SHROOM2 and RhoA-ROCK pathway interactions using immunofluorescence?

When investigating SHROOM2 and RhoA-ROCK pathway interactions via immunofluorescence, researchers must incorporate a comprehensive set of controls. First, include biological validation controls: SHROOM2 knockdown cells using validated shRNAs alongside rescue experiments with shRNA-resistant constructs to confirm specificity of observed phenotypes . Second, implement pharmacological controls: parallel treatments with ROCK inhibitor Y-27632 at 10μM to distinguish ROCK-dependent and independent functions, and RhoA activators (e.g., lysophosphatidic acid) or inhibitors to modulate upstream signaling . Third, use cytoskeletal visualization controls: co-stain with phalloidin to visualize F-actin organization, anti-Vinculin antibodies to mark focal adhesions, and phospho-MLC2 antibodies to assess ROCK activity . Fourth, incorporate subcellular fractionation controls: compare membrane versus cytosolic fractions in parallel Western blots to biochemically validate SHROOM2-dependent ROCK membrane recruitment observed in imaging . Fifth, implement technical fluorescence controls: single-stained samples for each fluorophore to establish spectral parameters, secondary-only controls to assess non-specific binding, and isotype controls matched to the SHROOM2 antibody. Sixth, include phenotypic validation controls: parallel analysis of epithelial markers (E-cadherin, desmoplakin) and mesenchymal markers (N-cadherin, vimentin) to correlate SHROOM2-ROCK interactions with functional outcomes . These systematic controls ensure that observed co-localization patterns and functional interactions between SHROOM2 and RhoA-ROCK pathway components represent genuine biological phenomena rather than technical artifacts.

How can researchers design experiments to study the differential roles of SHROOM2 in ROCK-dependent stress fiber formation versus ROCK-independent EMT suppression?

To dissect SHROOM2's dual roles in ROCK-dependent cytoskeletal organization versus ROCK-independent EMT suppression, researchers should implement a multi-dimensional experimental design. First, establish cellular systems with manipulated SHROOM2 expression: generate stable SHROOM2 knockdown lines using validated shRNAs, SHROOM2-overexpressing lines, and domain-specific mutants (SD1 and SD2 mutants that selectively disrupt F-actin or ROCK binding) . Second, implement a matrix-based experimental approach combining genetic and pharmacological perturbations: treat control cells, SHROOM2-depleted cells, and SHROOM2-overexpressing cells with or without ROCK inhibitor Y-27632, analyzing each condition for both cytoskeletal phenotypes and EMT markers . Third, employ high-resolution quantitative microscopy to simultaneously assess stress fiber formation (phalloidin staining), focal adhesion assembly (Vinculin immunostaining), and EMT marker expression/localization (E-cadherin, N-cadherin) . Fourth, perform time-course analyses during EMT induction (e.g., TGF-β treatment) to track the temporal relationship between stress fiber disassembly and EMT marker changes in control versus SHROOM2-depleted cells. Fifth, utilize advanced molecular approaches including proximity ligation assays to detect direct SHROOM2-ROCK interactions in situ, and ChIP-seq to identify potential transcriptional mechanisms underlying ROCK-independent functions. Sixth, validate findings in relevant disease models such as comparing primary and metastatic tumor samples for correlations between SHROOM2 expression, stress fiber patterns, and EMT status . This comprehensive approach enables precise delineation of the mechanistic boundaries between SHROOM2's cytoskeletal regulatory functions and its broader role in suppressing epithelial-mesenchymal transitions.

What emerging methodologies could enhance the detection and functional analysis of SHROOM2 beyond conventional FITC-conjugated antibody approaches?

Emerging methodologies are poised to revolutionize SHROOM2 detection and functional analysis beyond traditional FITC-conjugated antibody approaches. First, CRISPR-based endogenous tagging systems enable visualization of native SHROOM2 dynamics through knock-in of fluorescent proteins or self-labeling enzyme tags (HaloTag, SNAP-tag), avoiding potential artifacts from antibody binding or overexpression . Second, super-resolution microscopy techniques including STED, STORM, and SIM offer nanoscale resolution of SHROOM2's association with cytoskeletal structures and junctional complexes, revealing previously undetectable organizational details. Third, proximity labeling approaches using APEX2 or BioID fused to SHROOM2 enable comprehensive in situ identification of its protein interaction network across different subcellular compartments. Fourth, advanced live-cell imaging techniques using optogenetic SHROOM2 variants allow precise spatiotemporal control of its activity, enabling real-time analysis of its cytoskeletal regulatory functions. Fifth, single-cell multiomics approaches combining transcriptomics with protein detection provide insights into the heterogeneity of SHROOM2 expression and its correlation with cell state transitions. Sixth, tissue clearing methods coupled with light-sheet microscopy enable whole-organ imaging of SHROOM2 distribution in complex 3D tissues and organoids. Seventh, synthetic biology approaches using engineered SHROOM2 variants with tunable binding properties can dissect the specific contributions of different protein domains to its diverse functions. These innovative methodologies will provide unprecedented insights into SHROOM2 biology beyond what conventional antibody-based approaches have revealed.

How can FITC-conjugated SHROOM2 antibodies be effectively incorporated into multi-parametric flow cytometry panels for analyzing complex cell populations?

Incorporating FITC-conjugated SHROOM2 antibodies into multi-parametric flow cytometry requires strategic panel design to maximize information while minimizing spectral overlap. Begin with a validated permeabilization protocol optimized for intracellular cytoskeletal proteins: fix cells with 4% paraformaldehyde for 15 minutes followed by permeabilization with 0.1% saponin rather than harsher detergents that might disrupt cytoskeletal structures . For panel design, position FITC-SHROOM2 strategically considering its expression level - SHROOM2 is typically expressed at moderate levels, making FITC (excitation 495nm, emission 519nm) appropriate despite its relatively lower brightness compared to newer fluorophores. Combine with markers for cell identity, differentiation state, and other RhoA-ROCK pathway components, using fluorophores with minimal spectral overlap such as APC (CD44, mesenchymal marker), PE-Cy7 (E-cadherin, epithelial marker), and BV421 (phospho-MLC2, ROCK activity indicator). Include essential compensation controls: single-stained samples for each fluorophore, fluorescence-minus-one (FMO) controls particularly for FITC-SHROOM2, and isotype controls matched to the SHROOM2 antibody concentration. For accurate quantification, include calibration particles to convert fluorescence intensity to molecules of equivalent soluble fluorochrome (MESF). Validate the panel using known positive controls (cell lines with high SHROOM2 expression) and negative controls (SHROOM2-knockdown cells established with validated shRNAs) . For analysis, implement advanced computational approaches such as viSNE or FlowSOM to identify cell subpopulations with distinctive SHROOM2 expression patterns and correlate with epithelial/mesenchymal marker profiles. This approach enables quantitative assessment of SHROOM2 expression heterogeneity within complex tissues and its relationship to cellular differentiation states.

What novel insights could be gained by combining FITC-conjugated SHROOM2 antibody staining with transcriptomic analysis in single-cell studies?

Integrating FITC-conjugated SHROOM2 antibody staining with single-cell transcriptomics offers unprecedented opportunities to unravel SHROOM2's complex regulatory networks and functional heterogeneity. This approach enables researchers to correlate SHROOM2 protein levels and localization patterns with comprehensive gene expression profiles at single-cell resolution, revealing insights impossible to obtain through bulk analysis. First, researchers can identify transcriptional signatures specifically associated with different SHROOM2 expression levels or subcellular localization patterns, potentially uncovering novel regulatory relationships beyond the known RhoA-ROCK axis . Second, this approach allows precise mapping of SHROOM2's relationship to EMT states by correlating its expression with the full spectrum of epithelial-mesenchymal transition markers rather than selected proteins, capturing intermediate or hybrid EMT states that may have distinct SHROOM2 dependencies . Third, trajectory inference analyses can reveal the temporal dynamics of SHROOM2 regulation during cell state transitions, identifying potential transcription factors that regulate its expression. Fourth, cell-type specific SHROOM2 functions can be defined by analyzing correlation patterns between SHROOM2 and gene expression modules across diverse cell populations in complex tissues. Fifth, spatial transcriptomics combined with SHROOM2 immunofluorescence enables contextualization of its expression within tissue architecture and microenvironmental niches. For implementation, researchers should use well-validated methods like CITE-seq with oligonucleotide-conjugated SHROOM2 antibodies or sequential immunofluorescence followed by in situ RNA sequencing. These integrative approaches will yield systems-level understanding of SHROOM2's role in coordinating cytoskeletal dynamics, cell adhesion, and cell state transitions across diverse physiological and pathological contexts.