NCOA4 Antibodies

Nuclear receptor coactivator 4 (NCOA4)

Improves the androgen receptor transcriptional activity in prostate cancer cells. Ligand-independent coactivator of the peroxisome proliferator-activated receptor (PPAR) gamma.

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Gene Information

Human
Gene Name:	NCOA4
Uniprot:	Q13772
Entrez:	8031

Family

Belongs to:
No superfamily

Alternative Names

Androgen receptor coactivator 70 kDa protein; Androgen receptor-associated protein of 70 kDa; ARA70RFGret fused; DKFZp762E1112; ELE1NCoA-4; nuclear receptor coactivator 4,70 kDa AR-activator; PTC3RET-activating gene ELE1; Ret-activating protein ELE1,70 kDa androgen receptor coactivator

Molecular Weight

Mass (kDA):
69.726 kDA

Genomic Context

Human
Location:	10q11.22
Sequence:	10; NC_000010.11 (46005088..46030714, complement)

Tissue Specificity

Widely expressed. Also detected in adipose tissues and in different cell lines. Isoform Beta is only expressed in testis.

Diseases

• Malignant Neoplasms
• Neoplasms
• Malignant Neoplasm Of Prostate
• Carcinoma
• Thyroid Neoplasm
• Prostatic Neoplasms
• Carcinoma, Papillary
• Malignant Neoplasm Of Thyroid
• Papillary Thyroid Carcinoma
• Cell Transformation, Neoplastic
• Neoplasms, Radiation-induced
• Malignant Paraganglionic Neoplasm
• Neoplasm Metastasis

• Malignant Neoplasm Of Breast
• Mammary Neoplasms
• Hyperplasia
• Adenoma
• Ret/ptc Rearrangement
• Neoplasm Invasiveness
• Carcinogenesis

Pathways

• Cell Proliferation
• Cell Growth
• Receptor Transactivation
• Cell Cycle
• Pathogenesis
• Regulation Of Gene Expression
• Interphase
• Localization
• Reproductive System Development
• Spermatogenesis
• Cell Division
• System Development
• Organ Development

• Angiogenesis
• Secretion
• Proteolysis
• Cytokine Secretion
• Mammary Gland Development
• Vasculogenesis
• Liver Development

PTMs

• Phosphorylation

• Acetylation

For details, visit:
bosterbio.com/bosterbio-gene-info-cards/NCOA4

Best Uses Of The NCOA4 Marker

The NCOA4 marker, a cytokine, is produced by the kidneys of humans. Boster Bio is a well-known company that produces high quality antibodies as well as ELISA products. Recently, they have also expanded their product line to include PCR-related molecular biology products. Boster Bio offers many services to suit scientists' needs. They offer technical support and customer service 24 hours a day.

A supersensitive ECL solution was used to detect protein bands

To detect protein bandings in the LB membranes using a supersensitive ECL reagent, The supersensitive ECL reagent was used to detect the Boster Bio bands. The bovine IgG–HRP of anti-goat was added to PBS pH 7.4 with 0.1% BSA. Protein bands were visualized using Image Lab software.

The proteins from the pooled serum were separated by 8% sDS-PAGE, and transferred to PVDF membranes or NC membranes. The bound proteins were stained using different antibodies. Densitometry and scanning were used to determine the intensity of staining. The bands of two glycoproteins could be detected using one sample of each type. The NC membrane has a higher re-probing ability than the PVDF membrane.

The biliary exosomes contain more protein bands compared to the liver and gallbladder, which are largely enriched in exosomal markers. LAMP-1 relative expression, CD63, HSP-70, and CD63 were higher in the liver and the biliary exosomes than they were in gallbladder. This indicates that there is a significant enrichment of these exosomal marks in the liver and biliary exosomes.

Boster Bio's Supersensitive ECL Substrate Kit was created for the detection of proteins within a range of 23pg to 187ng per band. This enhanced chemiluminescent substrate can be used with both film and CCD imaging. Its high sensitivity allows for repeated exposures without worrying about losing data due poor background detection. In addition, Boster Bio protein bands were detected using a supersensitive ECL solution.

The primary and second antibodies were diluted in ECL reagent 5-10x more than usual. The usual dilution range is between 1/20,000 and 1/100,000. Some modifications were needed. After determining the dilution range, the membrane was rinsed with MilliQ or double-distilled waters. It is important for membranes to be rinsed with water to dry faster than those rinsed using the wash buffer.

As with any other enzyme-linked immunosorbent assay, an electrochemiluminescence (ECL) detector is used to detect proteins. It is a powerful method of detecting proteins within complex biological samples. ECL solutions are widely applicable to many applications. The ECL method is simple and effective for detecting trace biomarkers in biological samples because of its versatility.

Quantifications of protein bands intensities were performed using ImageJ software

ImageJ, a software tool that measures the intensity of protein bands, was used in the analysis of EMSA Gels. ImageJ allows you to quantify the intensities of selected peak peaks based upon their protein concentration and total volume. The software tool also allows you to calculate the binding constant of a protein. For more information on how to use ImageJ software for quantitative analysis of EMSA gels, read the paper we wrote in this series.

We used Kodak Biomax MR film with minimum and maximum OD values respectively of 0.06 and 1.6. Microtek Bio-5000 scanner was used to scan autoradiographs at 600 dpi resolution. DNA band was placed at the bottom of the image and was labeled as band 1. ImageJ software displayed gray values that correspond to the relative concentrations for each protein band.

The relative abundance of each band was calculated by enclosing each one in a regional of interest (ROI). This region is centered on the band at the molecular weight that corresponds to the target protein. To ensure proper background signal subtraction, we also included overlapping bands. We calculated the mean intensity of each band in the interest region and presented the results as a table.

The Straight Line Selection tool is available in the toolbar for ImageJ software. We use this tool to draw a line across each peak's base and select the first area of interest. From there, we identified the point at which the peak should be ended and where to begin the background noise. The Wand tool was used to highlight the peak, which was then labeled. After these steps were completed we saved the data in excel and exported it to our spreadsheet.

You should choose the lane that has the highest density. ImageJ assumes vertical orientation of the lanes, but individual bands are vertical. ImageJ's histogram will highlight only the first lane. This may create confusion during analysis. We recommend that you highlight the lane with the highest density.

The EMSA band intensity can be divided into multiple bands with a smear. Protein-DNA complexes are responsible for bands whose intensity changes according to the concentrations of protein bands. Bands that are almost constant in intensity are likely to be contamination. This is single-strandedDNA (ssDNA). This type of DNA migrates slowly, compared to its corresponding DNA.

PdtaA induces ferritinophagy

Autophagy is an important cellular function, but some cells also start ferritinophagy. Ferroptosis occurs when intracellular iron is increased and lipid peroxidation results. Researchers used knockdown of NCOA4 to examine the role PdtaA had in ferritinophagy. The mechanism involved is not yet fully understood, but is now considered a model for its regulation.

Inhibition of the xC-system, the autophagy receptor NCOA4, which regulates intracellular iron homeostasis, is essential for ferritinophagy. NCOA4 binds and transports ferritin to its pre-autophagosome structure. The ferritin gets degraded upon fusion between the autophagosome lysosome. This releases iron into the cells. Ferritinophagy can be important for iron-dependent cells death. Excessive iron can cause the formation of reactive oxygen compounds (ROS), that could be linked with neurodegeneration and oxidative stresses.

Previous studies on this pathway suggested that PdtaA's overexpression could inhibit ferritinophagy. A knockdown of NCOA4/p53 could decrease the effects of PdtaA's influence on the expression these proteins. When NCOA4 was blocked, however, siRNA-mate had a less significant effect on ferritinophagy in mice.

This study supports the notion that PdtaA promotes Ferritinophagy via regulation of NDRG1, a gene also implicated with EMT regulation. PdtaA induces ferroptosis by knocking down NDRG1. Moreover, knockdown of NDRG1 inhibited ferroptosis by upregulating Gpx4 and xCT, which were previously linked to ferroptosis.

PdtaA is also known to inhibit EMT by a mechanism that includes ROS production. N-acetyl L cysteine was used to study the role ROS plays in PdtaA's inhibition of EMT. Moreover, knockdown of NCOA4 completely neutralized the PdtaA effect on Bax, Bcl-2, and caspase-8.

PdtaA could also be observed to inhibit cell growth. PdtaA inhibited cell proliferation as evidenced by a decrease of Ki-67 levels and a significant rise in vimentin. PdtaA treatment also inhibited cell growth and significantly reduced lipid peroxidation. These results suggest that PdtaA plays crucial role in EMT.

In a 24-well plate, HepG2 cells were grown. The cells were fixed with 4% paraformaldehyde in PBS at 37degC. Each well was then incubated at 37°C for 24 h with fresh medium containing various concentrations PdtaA. The cells were then left to incubate at 37°C for 48 h in a humidified area. The cells were then fixed by 70% ethanol. In the absence of cell death, the cells were stained with 50 mg/ml PI.

NCOA4 is an transcription factor that mediates ferritinophagy. It was discovered first as a weak nuclear coactivator. Lodish et al. discovered that NCOA4 activated thyroid hormone by recruiting it into chromatin regions and transcriptions which are abundant during erythropoiesis. It has been shown that NCOA4 regulates DNA replication origin activation. It interacts avec minichromosome protein 7 protein.