rcc2 Antibody

What is RCC2 and what cellular functions does it regulate?

RCC2, also known as TD60, is a guanine nucleotide exchange factor (GEF) that is active on RalA, a small GTPase. RCC2 and RalA are both essential for proper kinetochore-microtubule function in early mitosis . RCC2 is a multifunctional protein that affects cellular processes by regulating the activity of small GTPases, such as RAC1 and RALA. It is required for normal progression through the cell cycle, both during interphase and mitosis. RCC2 is essential for the presence of normal levels of MAD2L1, AURKB, and BIRC5 on inner centromeres during mitosis, and for normal attachment of kinetochores to mitotic spindles . Additionally, it functions in the organization of the microtubule cytoskeleton in interphase cells.

What are the recommended applications for RCC2 antibodies?

RCC2 antibodies can be employed in multiple experimental techniques, with varying dilution requirements:

It is important to note that antibody performance is sample-dependent, and each reagent should be titrated in your specific testing system to obtain optimal results .

What is the molecular weight of RCC2 protein?

RCC2 protein has a calculated molecular weight of 56 kDa based on its amino acid sequence (522 amino acids) . The observed molecular weight in SDS-PAGE analysis is approximately 56-60 kDa . This slight variation in observed weight may be due to post-translational modifications or differences in experimental conditions.

What species reactivity do commercial RCC2 antibodies exhibit?

When selecting an antibody for your experiments, it's crucial to choose one with demonstrated reactivity to your species of interest. Cross-checking the reactivity data with your experimental design will help ensure successful detection.

How is RCC2 involved in cancer development and progression?

RCC2 has been demonstrated to play significant roles in multiple cancer types:

In ER-positive breast cancer:

• Western blotting and immunohistochemistry have detected significantly increased expression of RCC2 in ER+ breast tumor tissues compared with breast fibroadenoma samples .

• Inhibition of RCC2 expression decreases cell migration and stimulates apoptosis in MCF-7 cells (an ER+ breast cancer cell line) .

• Conversely, overexpression of RCC2 stimulates cell migration and inhibits apoptosis .

• In mouse models, inhibition of RCC2 expression significantly decreased breast tumor growth and IL-6 levels in tumor-bearing mice .

In colorectal cancer:

• RCC2 has been identified as a potential biomarker for colorectal cancer .

These findings suggest that RCC2 functions as a tumor-promoting factor in certain cancers, particularly in ER+ breast cancer, where it appears to influence multiple aspects of tumor cell behavior including migration, survival, and growth.

What signaling pathways does RCC2 interact with in cancer cells?

RCC2 interacts with several key signaling pathways that contribute to cancer progression:

• IGF1 pathway: PCR array analysis has demonstrated that inhibiting RCC2 expression significantly decreases the expression of IGF1 (insulin-like growth factor 1), a well-known tumor-enhancing gene, in MCF-7 cells. Conversely, overexpressing RCC2 increases IGF1 expression levels .

• TWIST1 pathway: Similarly, RCC2 regulates TWIST1 expression, with inhibition of RCC2 decreasing TWIST1 levels and RCC2 overexpression increasing TWIST1 levels . TWIST1 is an important transcription factor involved in epithelial-mesenchymal transition (EMT) and cancer metastasis.

• Estrogen signaling: In ER+ breast cancer cells, estradiol-17β has been shown to suppress apoptosis, stimulate cell proliferation and migration, and increase RCC2, IGF1, and TWIST1 expression . This suggests a potential regulatory relationship between estrogen signaling and RCC2 function.

• RAC1 signaling: RCC2 interferes with the activation of RAC1 by guanine nucleotide exchange factors. It prevents accumulation of active, GTP-bound RAC1, and suppresses RAC1-mediated reorganization of the actin cytoskeleton and formation of membrane protrusions .

• RALA signaling: RCC2 functions as a guanine nucleotide exchange factor (GEF) for RALA, activating this small GTPase which plays roles in various cellular processes including vesicle trafficking and cytokinesis .

What are the technical considerations for optimizing RCC2 antibody use in immunohistochemistry?

For optimal RCC2 antibody performance in immunohistochemistry, consider the following technical aspects:

Antigen retrieval methods:

• Primary recommendation: Use TE buffer pH 9.0 for antigen retrieval

• Alternative method: Citrate buffer pH 6.0 may also be effective

Tissue compatibility:
RCC2 antibody 16755-1-AP has shown positive IHC detection in:

• Human tissues: colon cancer tissue, ovary tumor tissue, stomach cancer tissue

• Animal tissues: mouse testis tissue, rat colon tissue, rat testis tissue

Dilution optimization:

• Starting dilution range: 1:250-1:1000

• Each specific tissue type may require optimization within this range

• Consider performing a dilution series test on your specific sample type

Controls:

• Include positive controls from the validated tissue types listed above

• Use negative controls (primary antibody omission or isotype control)

• Consider using tissues known to express different levels of RCC2 for calibration

Detection systems:

• Compatible with standard immunoperoxidase and fluorescent secondary detection systems

• Signal amplification systems may be beneficial for detecting lower expression levels

How do experimental results from RCC2 inhibition studies inform potential therapeutic applications?

In vitro and in vivo studies of RCC2 inhibition have yielded several significant findings with therapeutic implications:

• Anti-migratory effects: Inhibiting RCC2 expression decreases cell migration in MCF-7 cells, suggesting that targeting RCC2 might reduce cancer cell invasiveness and metastatic potential .

• Pro-apoptotic effects: RCC2 inhibition stimulates apoptosis in MCF-7 cells, indicating that RCC2 normally functions to promote cancer cell survival .

• Tumor growth inhibition: In mouse models, inhibition of RCC2 expression significantly decreased breast tumor growth, demonstrating in vivo efficacy of RCC2 targeting .

• Cytokine modulation: RCC2 inhibition reduced IL-6 levels in tumor-bearing mice, suggesting effects on the tumor microenvironment and inflammatory signaling .

• Downstream pathway regulation: RCC2 inhibition significantly decreases expression of IGF1 and TWIST1, two well-established tumor-promoting genes . This indicates that RCC2 targeting may disrupt multiple oncogenic pathways simultaneously.

• Estrogen signaling interaction: Since estradiol-17β increases RCC2 expression, and RCC2 promotes cancer cell survival and migration, RCC2 may represent a mediator of estrogen's pro-tumorigenic effects in ER+ breast cancer . This suggests potential synergistic benefits of combining RCC2 inhibition with anti-estrogen therapies.

These findings collectively suggest that RCC2 may represent a promising therapeutic target, particularly in ER+ breast cancer, with potential for affecting multiple cancer hallmarks including survival, migration, and growth.

What experimental controls should be included when using RCC2 antibodies in various applications?

Additionally, when studying RCC2 in cancer contexts, consider including:

• Normal adjacent tissue controls to compare with tumor samples

• A panel of cancer cell lines with varying RCC2 expression levels

• Positive and negative controls for subcellular localization experiments (RCC2 should show both nuclear and cytoplasmic distribution with enrichment at kinetochores during mitosis)

• siRNA/shRNA knockdown validation to confirm antibody specificity and establish knockdown efficiency for functional studies

What are the recommended protocols for using RCC2 antibodies in Western blotting?

Sample Preparation:

• Prepare cell lysates in a standard lysis buffer containing protease inhibitors

• Determine protein concentration using a standard assay (BCA, Bradford)

• Prepare samples with 20-50 μg total protein per lane

• Add reducing sample buffer and heat at 95°C for 5 minutes

Electrophoresis and Transfer:

• Resolve proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

Antibody Incubation:

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Incubate with primary RCC2 antibody at the appropriate dilution:

  • 1:1000-1:4000 for 16755-1-AP

  • 1:500-1:2000 for STJ115071

  • 1:1000 for #3667

• Incubate overnight at 4°C or 2-3 hours at room temperature

• Wash 3-5 times with TBST

• Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

• Wash 3-5 times with TBST

Detection:

• Apply ECL substrate and expose to film or use a digital imaging system

• Expected RCC2 band at approximately 56-60 kDa

Troubleshooting:

• If multiple bands appear, increase antibody dilution or reduce sample loading

• If no band appears, decrease antibody dilution, increase exposure time, or confirm sample contains RCC2 (use positive control)

• High background may indicate insufficient blocking or washing

How can researchers effectively use RCC2 antibodies to study its role in mitosis?

RCC2 plays crucial roles during mitosis, particularly in kinetochore-microtubule function . To effectively study these functions:

Immunofluorescence for Mitotic Localization:

• Culture cells on glass coverslips or chamber slides

• Fix cells with 4% paraformaldehyde (10 minutes) or ice-cold methanol (5 minutes)

• Permeabilize with 0.2% Triton X-100 (only if using paraformaldehyde)

• Block with 3-5% BSA in PBS for 30-60 minutes

• Incubate with RCC2 antibody at 1:200 dilution overnight at 4°C

• Wash 3-5 times with PBS

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Co-stain with anti-tubulin antibody to visualize mitotic spindles

• Counterstain DNA with DAPI or Hoechst

• Mount and image using confocal microscopy

Cell Synchronization for Mitotic Studies:

• Synchronize cells at G1/S boundary using double thymidine block or at G2/M using nocodazole

• Release cells and collect at various timepoints covering mitotic progression

• Confirm mitotic stages by microscopy or flow cytometry

• Analyze RCC2 protein levels, modifications, or localization across the cell cycle

RCC2 Functional Studies in Mitosis:

• Use siRNA or shRNA to deplete RCC2 (validated sequences from literature )

• Analyze mitotic progression using live-cell imaging

• Quantify mitotic defects (misaligned chromosomes, multipolar spindles, etc.)

• Perform rescue experiments with wild-type or mutant RCC2 constructs

• Co-immunoprecipitate to identify mitotic binding partners

What considerations are important when designing RCC2 knockdown experiments?

When designing RCC2 knockdown experiments for functional studies, consider the following:

siRNA/shRNA Design:

• Target regions of high sequence conservation if studying RCC2 across multiple species

• Avoid regions with sequence similarity to other genes

• Design multiple siRNAs targeting different regions of RCC2 mRNA

• Include non-targeting control siRNA/shRNA

Validated siRNA Sequences:
Previous studies have successfully used RCC2 siRNA in MCF-7 cells for functional studies . Consider using these validated sequences as a starting point.

Knockdown Verification:

• Confirm knockdown efficiency by Western blot (protein level) and qRT-PCR (mRNA level)

• Time course analysis to determine optimal timepoint for functional assays

• Typically 48-72 hours post-transfection for maximum knockdown effect

Functional Readouts:
Based on known RCC2 functions, consider these assays:

• Cell migration (wound healing, transwell migration)

• Apoptosis (flow cytometry for Annexin V, caspase activation)

• Cell proliferation (CCK-8 assay, BrdU incorporation)

• Gene expression analysis (focus on IGF1 and TWIST1)

• Cytokine production (especially IL-6)

• Cell cycle progression (flow cytometry, live cell imaging)

• Microtubule organization (immunofluorescence)

Rescue Experiments:

• Express siRNA-resistant RCC2 to confirm specificity of observed phenotypes

• Consider domain-specific mutants to dissect structure-function relationships

How does estrogen signaling interact with RCC2 function in ER+ breast cancer?

Research has revealed important interactions between estrogen signaling and RCC2 in ER+ breast cancer:

• Estrogen upregulates RCC2 expression: Estradiol-17β treatment increases RCC2 expression in MCF-7 cells, suggesting that RCC2 is an estrogen-responsive gene .

• Parallel cellular effects: Both estradiol-17β and RCC2 overexpression produce similar cellular phenotypes:

  • Suppression of apoptosis

  • Stimulation of cell proliferation

  • Enhancement of cell migration

• Downstream effectors: Estradiol-17β increases expression of RCC2, IGF1, and TWIST1 in MCF-7 cells, suggesting a potential regulatory cascade .

• Functional relationship: siRNA-mediated inhibition of RCC2 can at least partially block the effects of estradiol-17β on cell apoptosis, suggesting that RCC2 is a mediator of estrogen's anti-apoptotic effects .

Experimental Approaches to Study Estrogen-RCC2 Interactions:

• Time-course analysis: Treat MCF-7 cells with estradiol-17β and monitor RCC2 expression over time (0, 6, 12, 24, 48 hours)

• Dose-response studies: Treat cells with varying concentrations of estradiol-17β to determine threshold for RCC2 induction

• ER antagonist studies: Pre-treat cells with tamoxifen or fulvestrant before estradiol-17β addition to confirm ER-dependency

• ChIP assays: Determine if ERα directly binds to RCC2 promoter regions

• Reporter assays: Clone RCC2 promoter into luciferase reporter to quantify transcriptional activation by estrogen

• RCC2 knockdown effects: Compare estradiol-17β effects in control vs. RCC2-depleted cells

These experimental approaches can help elucidate the mechanistic relationship between estrogen signaling and RCC2 function in breast cancer.

What emerging applications of RCC2 antibodies show promise for cancer research?

Several emerging applications of RCC2 antibodies show significant potential for advancing cancer research:

• Prognostic biomarker development: Given RCC2's elevated expression in breast and colorectal cancers , RCC2 immunohistochemistry might be developed into a clinically useful prognostic tool. Research could focus on correlating RCC2 expression levels with patient outcomes across multiple cancer types and stages.

• Therapeutic response prediction: Studies could investigate whether RCC2 expression levels correlate with response to specific therapies, particularly in ER+ breast cancer where RCC2 appears to mediate estrogen effects .

• Liquid biopsy development: Research into detecting RCC2 protein or autoantibodies in patient serum could potentially lead to minimally invasive diagnostic or monitoring tools.

• Single-cell analysis: Using RCC2 antibodies in mass cytometry or single-cell Western blotting could reveal heterogeneity in RCC2 expression within tumors and help identify particularly aggressive subpopulations.

• Drug discovery applications: RCC2 antibodies could be employed in high-throughput screening assays to identify compounds that modulate RCC2 expression or function, potentially leading to novel therapeutic agents.

• Mechanistic studies of RCC2 in cancer stem cells: Investigating RCC2's role in cancer stem cell maintenance could provide insights into therapy resistance and disease recurrence.

• Combination therapy rational design: Understanding how RCC2 expression affects response to standard therapies could inform rational combination approaches, particularly in cancers where RCC2 is overexpressed.

What outstanding questions remain about RCC2's role in cancer biology?

Despite progress in understanding RCC2's functions, several important questions remain unresolved:

• Upstream regulation: While estrogen has been shown to increase RCC2 expression in ER+ breast cancer cells , the complete transcriptional and post-transcriptional regulatory mechanisms controlling RCC2 expression in normal and cancer cells remain poorly understood.

• Protein interactions: The complete interactome of RCC2 in different cancer contexts has not been fully characterized. Identifying cancer-specific binding partners could reveal novel therapeutic vulnerabilities.

• Post-translational modifications: How phosphorylation, ubiquitination, or other modifications affect RCC2 function in cancer cells is not well established.

• Cancer-specific functions: Whether RCC2 has fundamentally different functions in cancer cells compared to normal cells needs further investigation.

• Metabolic connections: Potential roles for RCC2 in regulating cancer metabolism have not been extensively explored.

• Immune interactions: Whether RCC2 expression affects tumor-immune interactions or immunotherapy response remains unknown.

• Therapy resistance: RCC2's potential role in developing resistance to standard cancer therapies deserves investigation, particularly in breast cancer where it promotes survival and migration.

• Cross-talk with other oncogenic pathways: While connections to IGF1 and TWIST1 have been established , the full extent of RCC2's integration with other cancer signaling networks requires further study.

Addressing these questions will require innovative experimental approaches and may open new avenues for therapeutic intervention in RCC2-expressing cancers.