esco2 Antibody

What is ESCO2 and where is it primarily localized in cells?

ESCO2 is an acetyltransferase involved in sister chromatid cohesion during mitotic S-phase. Immunofluorescence studies reveal that ESCO2 predominantly localizes to chromosomes, particularly during germinal vesicle breakdown (GVBD) through metaphase II stages. The protein distributes along interstitial chromosome axes, extending over centromere regions and arm regions both proximal and distal to chiasmata. Additionally, ESCO2 can be found in the periphery of sister homologs, suggesting functions beyond chromosome cohesion .

How does ESCO2 expression differ between normal and cancer tissues?

ESCO2 expression is significantly elevated in various cancer tissues compared to matched normal tissues. Bioinformatic analyses using TCGA data have revealed that ESCO2 is upregulated in head and neck squamous cell carcinoma (HNSC) compared to normal tissues (P < 0.001). Analysis of 43 paired specimens confirmed significantly higher ESCO2 transcription levels in tumors than in paired-adjacent normal tissues . This differential expression pattern suggests ESCO2's potential utility as a pan-cancer biomarker .

What are the recommended positive and negative controls when using ESCO2 antibodies?

For positive controls, researchers should utilize cell lines known to express ESCO2, such as FaDu cells used in hypopharyngeal carcinoma studies . For negative controls, ESCO2-depleted samples generated through morpholino knockdown or shRNA approaches offer ideal comparison points. When validating specificity, western blotting should demonstrate a significant decrease in ESCO2 protein levels in knockdown samples compared to controls, as confirmed by both immunoblotting and immunofluorescence analysis .

How can ESCO2 antibodies be used to investigate its binding partners?

Co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS) is the recommended approach for identifying ESCO2 binding partners. This method has successfully revealed the interaction between ESCO2 and STAT1 in hypopharyngeal carcinoma cells . For verification of specific interactions, researchers should perform reciprocal Co-IP experiments using antibodies against both ESCO2 and the suspected binding partner, followed by immunoblotting. This approach helps avoid false positives and confirms the biological relevance of the interaction.

What methodologies are recommended for studying ESCO2's role in the spindle assembly checkpoint (SAC)?

To investigate ESCO2's involvement in SAC, researchers should employ a multi-method approach:

• Nocodazole treatment to induce metaphase I arrest, followed by quantification of checkpoint override rates in ESCO2-depleted versus control cells

• Immunofluorescent staining to visualize spindle morphologies (using anti-α-tubulin antibody) and chromosome alignment

• Cold-stable microtubule assays to assess kinetochore-microtubule attachment stability

Control experiments showed only ~13% of cells override the metaphase I arrest induced by nocodazole, while ESCO2-depleted cells showed a significantly increased override incidence (~31%), indicating SAC inactivation. These findings highlight that ESCO2 is critical for maintaining SAC activity .

How should researchers design experiments to investigate ESCO2's acetyltransferase activity?

To study ESCO2's acetyltransferase function, researchers should utilize in vitro acetylation assays. This involves:

• Expression and purification of recombinant ESCO2 (wild-type and catalytically inactive mutants like W530G)

• Incubation of purified ESCO2 with recombinant histone H4 in acetyltransferase assay buffer

• Analysis by western blotting using anti-H4K16ac antibody to detect acetylation

This approach has confirmed ESCO2's ability to acetylate H4K16 both in vivo and in vitro . For comprehensive analysis, researchers should include controls lacking Ac-CoA and compare wild-type ESCO2 with catalytically inactive mutants.

What are the optimal conditions for ESCO2 immunofluorescence staining?

For optimal ESCO2 immunofluorescence:

• Fix samples with 4% paraformaldehyde for 30 minutes

• Permeabilize with 0.5% Triton X-100 for 20 minutes

• Block with 1% BSA in PBS for 1 hour

• Incubate with primary ESCO2 antibody overnight at 4°C

• Counterstain with appropriate markers (e.g., PI for DNA visualization, α-tubulin for spindle structures, CREST for kinetochores)

This protocol has successfully demonstrated ESCO2's chromosomal localization during oocyte meiotic maturation . When visualizing kinetochore-microtubule attachments, combining ESCO2 antibody with CREST and α-tubulin antibodies provides comprehensive structural context.

What knockdown approaches are most effective for ESCO2 functional studies?

Two principal approaches have proven effective for ESCO2 depletion:

• Morpholino-based knockdown: Using Esco2-targeting morpholino (5′-TCTTGGAGTACAAGTTGCCATCATC-3′) microinjected into cells at 1mM working concentration. This approach requires incubation for 20 hours to facilitate morpholino-mediated inhibition of mRNA translation .

• shRNA-mediated silencing: Utilizing shRNA constructs targeting ESCO2 (e.g., shESCO2-1, shESCO2-2). This approach has demonstrated significant suppression of ESCO2 expression in cancer cell lines like FaDu .

Validation of knockdown efficiency via western blot and immunofluorescence is essential before proceeding with functional studies.

How does ESCO2 influence cancer cell proliferation and viability?

ESCO2 significantly impacts cancer cell proliferation and viability. Experimental data shows that ESCO2 depletion using shRNA constructs (shESCO2-1, shESCO2-2) suppresses tumor cell growth beginning from day 2 of treatment. Notably, introducing shESCO2-1 has been shown to completely abrogate FaDu cell growth in hypopharyngeal carcinoma models. Cell viability assays confirm that HPC viability is significantly impaired by ESCO2 depletion, with inhibitory effects observable as early as 2 days after experiment initiation .

What is the relationship between ESCO2 expression and clinical parameters in cancer patients?

How can ESCO2 antibodies help identify potential therapeutic targets in cancer research?

ESCO2 antibodies can reveal critical protein interactions that represent potential therapeutic targets. Co-IP/MS experiments using ESCO2 antibodies have identified STAT1 as an important binding partner in HPC cells. Functional studies confirmed that STAT1 overexpression compromises ESCO2-mediated suppressive effects on HPC cell proliferation, viability, and migration, suggesting the ESCO2-STAT1 axis represents a potential therapeutic target . By employing ESCO2 antibodies in similar interaction studies across different cancer types, researchers can identify cancer-specific binding partners and pathways for targeted intervention.

What are the recommended procedures for analyzing chromosomal abnormalities resulting from ESCO2 dysfunction?

To evaluate chromosomal abnormalities induced by ESCO2 dysfunction, researchers should implement:

• Cold-stable microtubule assays: Expose cells to cold treatment to induce depolymerization of microtubules not attached to kinetochores, then immunolabel with:

  • CREST antibody to visualize kinetochores

  • Anti-α-tubulin-FITC antibody to visualize microtubule fibers

  • DNA counterstain (e.g., Hoechst)

• Chromosome spreading and counting: To quantify aneuploidy rates:

  • Harvest cells at metaphase II stage

  • Prepare chromosome spreads

  • Count chromosome numbers in at least 30-50 cells per condition

Research has demonstrated that ESCO2 depletion substantially increases the proportion of impaired kinetochore-microtubule attachments (78.59 ± 1.81% vs. 29.42 ± 2.01% in controls) and significantly increases aneuploidy rates (71.19 ± 3.41% vs. 24.13 ± 1.56% in controls) .

How can researchers use ESCO2 antibodies in xenograft models to study tumor progression?

For xenograft studies incorporating ESCO2 antibody applications:

• Establish cell line-derived xenografts using control and ESCO2-depleted cells (e.g., shCtrl, shESCO2-1)

• Subcutaneously inoculate cells in nude mice (typically 4.0 × 10^6 cells per mouse)

• Monitor tumor growth over time

• At study endpoint, harvest tumors for:

  • Immunohistochemistry using ESCO2 antibodies to confirm knockdown maintenance

  • Analysis of proliferation markers

  • Assessment of ESCO2 binding partners (e.g., STAT1) via co-immunostaining

This approach has confirmed that ESCO2 depletion inhibits tumor growth in vivo, supporting in vitro findings and validating ESCO2 as a potential therapeutic target .