FOXRED2 Antibody

What is FOXRED2 and what cellular functions does it perform?

FOXRED2 (FAD-dependent oxidoreductase domain-containing protein 2) is a flavoprotein primarily involved in endoplasmic reticulum associated degradation (ERAD). It functions by binding non-native proteins in the endoplasmic reticulum and targeting them to the ubiquitination machinery for subsequent degradation . FOXRED2 plays crucial roles in maintaining mitochondrial function and redox regulation. Recent research has revealed that FOXRED2 is highly expressed in various human tumors, including cutaneous malignant melanoma (CMM), non-small cell lung cancer, and colorectal cancer . Dysregulation of FOXRED2 has been linked to various diseases, including neurodegenerative disorders and cancer .

What are the validated applications for FOXRED2 antibody?

FOXRED2 antibodies have been validated for several experimental applications, including:

• Enzyme-Linked Immunosorbent Assay (ELISA) with recommended dilutions of 1:2000-1:10000

• Immunohistochemistry (IHC) with recommended dilutions of 1:20-1:200

• Western blot analysis

• Immunofluorescence techniques

Each application requires specific optimization depending on the experimental design and tissue/cell types being studied.

What is the cellular localization of FOXRED2?

FOXRED2 is primarily localized in the endoplasmic reticulum lumen, consistent with its role in endoplasmic reticulum associated degradation (ERAD) . When using immunohistochemistry techniques, successful staining typically shows FOXRED2 expression patterns as red particle deposition in the cytoplasm and cell membrane . Understanding this localization pattern is essential for correctly interpreting immunostaining results in various experimental contexts.

How can researchers optimize FOXRED2 antibody use in melanoma studies?

Based on recent findings regarding FOXRED2's role in melanoma, researchers should consider the following optimization approaches:

• Tissue sample selection: Include normal skin, nevus, and melanoma tissues for comparative analyses, as FOXRED2 expression shows significant differences between these tissue types. In one study, positivity rates were 0% in normal control tissues, 0% in nevus tissues, and 89.5% in melanoma tissues .

• Cell line selection: Consider using both primary melanoma (e.g., A375) and metastatic melanoma (e.g., A2058) cell lines, as they exhibit different FOXRED2 expression levels and functional impacts. Studies have shown that A2058 cells demonstrate higher FOXRED2 gene expression levels compared to A375 cells .

• Experimental timing: For siRNA knockdown experiments, optimize the reaction timing of siRNA transfection and protein expression at 48-72 hours, as this timeframe has been confirmed in pre-experiments to be optimal .

• Expression analysis methods: Combine multiple methods (RT-qPCR, Western blot, immunohistochemistry) for comprehensive expression analysis, as mRNA and protein levels may not always positively correlate .

How should I interpret contradictory FOXRED2 expression data between mRNA and protein levels?

Researchers have observed situations where FOXRED2 mRNA and protein expression levels in cells are not positively correlated . When confronting such contradictions:

• Consider protein regulation mechanisms: Post-transcriptional and post-translational modifications may affect protein levels independently of mRNA expression.

• Temporal dynamics: Protein expression may lag behind mRNA expression, creating apparent discrepancies at specific time points.

• Spatial considerations: FOXRED2 protein levels may be regulated independently of the spatiotemporal interval .

• Verification approach: Use multiple techniques (RT-qPCR, Western blot, immunohistochemistry) and biological replicates to confirm findings.

• Control selection: Include appropriate housekeeping genes and proteins as internal controls to normalize expression data.

When analyzing such data, report both mRNA and protein expression patterns while acknowledging potential regulatory mechanisms that might explain the discrepancies.

What are the key considerations when designing FOXRED2 knockdown experiments?

Based on previous successful knockdown studies, researchers should consider:

• siRNA design: Multiple siRNA sequences should be tested to identify the most effective one. In previous studies, researchers tested three different siRNAs (si1, si2, and si3) targeting FOXRED2 .

• Knockdown verification: Verify knockdown efficiency at both mRNA level (using RT-qPCR) and protein level (using Western blot) to ensure complete evaluation of knockdown effects .

• Functional assays: Include multiple assays to assess the effects of FOXRED2 knockdown:

  • CCK-8 assay for cell proliferation

  • Scratch-wound healing assay for migration

  • Transwell assays for invasion and migration

  • Flow cytometry for apoptosis analysis

• Time-course analysis: Monitor the effects of FOXRED2 knockdown over time, as different cellular processes may be affected at different time points after knockdown.

How can I effectively analyze FOXRED2's prognostic value in cancer studies?

To analyze FOXRED2's prognostic value in cancer studies:

The table below summarizes a representative analysis of FOXRED2 expression in different tissue types:

Table 1: FOXRED2 expression in different tissue types. Red particle deposition in the cytoplasm and cell membrane was considered to indicate positive findings.

What are the recommended protocols for FOXRED2 immunohistochemistry?

Based on successful experimental approaches, the following protocol is recommended for FOXRED2 immunohistochemistry:

• Antibody selection: Use rabbit anti-human FOXRED2 monoclonal antibody at 1:200 dilution or the FOXRED2 polyclonal antibody (PACO52534) at 1:20-1:200 dilution for optimal results .

• Secondary antibody: Apply secondary antibody conjugated with HRP followed by AEC Peroxidase Substrate Kit (e.g., Solarbio, Inc. cat. A2010) and hematoxylin counterstaining .

• Positive result interpretation: Consider red particles deposited on the cytoplasm and cell membrane as positive staining .

• Controls: Include normal skin and nevus tissues as negative controls, as they typically show negative FOXRED2 expression .

• Quantification: Score results systematically, categorizing samples as positive or negative based on established criteria. Results should be evaluated by at least two independent investigators to ensure reliability .

How can I troubleshoot inconsistent results in FOXRED2 expression analysis?

When encountering inconsistent results:

• Cross-validation: Use multiple techniques (RT-qPCR, Western blot, immunohistochemistry) to verify expression patterns.

• Cell line heterogeneity: Consider that different melanoma cell lines (e.g., A2058 vs. A375) show different FOXRED2 expression patterns and may respond differently to experimental manipulations .

• Antibody validation: Verify antibody specificity using positive and negative controls. For the FOXRED2 antibody (PACO52534), human brain tissue has been successfully used for validation at a dilution of 1:100 .

• Sample size considerations: Be aware that small sample sizes may lead to statistically insignificant results despite apparent trends, particularly for correlation analyses with clinicopathological features .

• Technical factors: Consider variables such as tissue fixation time, processing methods, and antibody lot-to-lot variations that might affect staining consistency.

What are the best experimental designs for studying FOXRED2's role in tumor progression?

Based on successful research approaches, consider the following experimental design:

• Multi-level analysis:

  • Bioinformatics analysis of FOXRED2 expression in databases like GEPIA2 and TIMER

  • Immunohistochemical analysis of patient samples

  • In vitro functional studies with cell lines

• Functional assays after FOXRED2 manipulation:

  • Proliferation: CCK-8 assay (significant inhibition observed in A2058 cells at p<0.01 and A375 cells at p<0.05 after FOXRED2 knockdown)

  • Migration: Scratch-wound healing assay (inhibition observed at p<0.01 in A2058 and p<0.001 in A375 cells)

  • Invasion and migration: Transwell assays

  • Apoptosis: Flow cytometry (increased apoptosis ratio observed at p<0.05 in A2058 and p<0.001 in A375 cells)

• Comparative approach: Compare primary tumor cells (A375) with metastatic cells (A2058) to understand FOXRED2's role in different stages of tumor progression .

• Molecular pathway analysis: Investigate interactions between FOXRED2 and its potential downstream targets to elucidate the molecular mechanisms behind its oncogenic effects.

What are the optimal dilution ratios for different FOXRED2 antibody applications?

Based on validated protocols, the following dilution ratios are recommended:

Table 2: Recommended FOXRED2 antibody dilution ranges for different applications.

Specific optimization may be required depending on the exact experimental conditions, tissue type, and detection method used.

How can I optimize FOXRED2 siRNA knockdown experiments?

Based on successful knockdown protocols:

• siRNA selection: Multiple siRNA sequences should be tested. Previous studies evaluated three different siRNAs (si1, si2, and si3), with no statistically significant differences in knockdown efficiency between them .

• Transfection timing: The optimal reaction timing for siRNA transfection and protein expression has been confirmed to be 48-72 hours in both A2058 and A375 melanoma cell lines .

• Efficiency verification:

  • At mRNA level: Verify knockdown using RT-qPCR with appropriate primers

  • At protein level: Confirm reduced expression via Western blot

• Controls: Include both blank (untreated) and negative control (scrambled siRNA) groups in all experiments to account for non-specific effects .

What are the important considerations for interpreting FOXRED2 immunohistochemistry results in clinical samples?

When interpreting clinical samples:

How might FOXRED2 research connect to broader oncology investigations?

The emerging research on FOXRED2 connects to broader oncology in several ways:

• Pan-cancer relevance: FOXRED2 appears to be a highly expressed pan-oncogene in various human tumors beyond melanoma, including non-small cell lung cancer and colorectal cancer .

• Prognostic biomarker development: Given the significant correlation between high FOXRED2 expression and lower survival rates in melanoma patients, further research could validate FOXRED2 as a prognostic biomarker across multiple cancer types .

• Therapeutic target exploration: The inhibition of cell proliferation, invasion, and migration, along with increased apoptosis observed after FOXRED2 knockdown, suggests its potential as a therapeutic target .

• Metastasis mechanisms: The significantly reduced invasion and migration ability of metastatic melanoma cells after FOXRED2 knockdown points to its involvement in metastatic processes, a critical area of cancer research .

What are the challenges in establishing correlations between FOXRED2 expression and clinical outcomes?

Researchers face several challenges:

• Sample size limitations: For rare cancers like cutaneous melanoma, obtaining sufficient sample sizes for statistically robust analyses is difficult. This may explain why some studies fail to find statistically significant correlations between FOXRED2 expression and clinicopathological features despite apparent trends .

• Sample stratification: Uneven distribution of samples across different disease stages can affect statistical analyses. For example, in one study, samples with <0.5 cm in Breslow thickness grading accounted for about 60% of the total patient volume, while samples with Breslow thickness >1.0 cm were only 2 cases .

• Biological variability: Heterogeneity in tumor samples, varying levels of lymphocyte infiltration, and other biological factors can introduce variability that complicates correlation analyses.

• Technical variability: Differences in tissue processing, staining protocols, and scoring systems across studies can make comparisons difficult.

Despite these challenges, there is evidence suggesting an increasing trend in the relationship between FOXRED2 expression and the clinicopathological features of cutaneous melanoma that may be related to tumor progression .