Screening and identification of proteins interacting with nucleostemin

AIM： To identify the proteins interacting with nucleostemin （NS）, thereby gaining an insight into the function of NS. METHODS： Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subseque...

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Published in:World journal of gastroenterology : WJG 2005-08, Vol.11 (31), p.4812-4814
Main Authors: Yang, Hai-Xia, Jin, Geng-Lin, Meng, Ling, Zhang, Jian-Zhi, Liu, Wen-Bin, Shou, Cheng-Chao
Format: Article
Language:English
Subjects:
Animals
Basic Research
Carrier Proteins - metabolism
Cercopithecus aethiops
Cloning, Molecular
COS Cells
Female
GTP-Binding Proteins
Humans
Nuclear Proteins - metabolism
Placenta - metabolism
Plasmids
Pregnancy
Recombinant Proteins - metabolism
交互作用
免疫沉淀反应
蛋白质
鉴别诊断
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Summary:AIM： To identify the proteins interacting with nucleostemin （NS）, thereby gaining an insight into the function of NS. METHODS： Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS： Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B （B56） （PPP2RSA） and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION： NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.
ISSN:1007-9327
2219-2840
DOI:10.3748/wjg.v11.i31.4812