[Screening of deformed epidermal autoregulatory factor 1-related functional proteins].
OBJECTIVE
To obtain HEK293T cell line which expresses deformed epidermal autoregulatory factor-1 (DEAF1) highly and stably, and to screen candidate proteins which can interact with DEAF1 by co-immunoprecipitation (Co-IP) and mass spectrometry (MS).


METHODS
Plasmids pEGFP-mDEAF1 and pEGFP-N1 were transfected into HEK293T and 2F-2B cell lines with TurboFect transfection reagent, respectively. The localization of DEAF1 was observed in the two kinds of cell lines by a fluorescence microscope. Plasmid p3×FLAG-mDEAF1 was transfected into HEK293T and 2F-2B cell lines, respectively. Non-transfected cells were used as a control group. The expression levels of DEAF1 mRNA and protein were detected by reverse transcription PCR, real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. Plasmid p3×FLAG-mDEAF1 was transfected into HEK293T cells, and then HEK293T cell line expressing stably DEAF1 was selected with G418 and identified by Western blotting, immunofluorescence and flow cytometry. Nuclear proteins of HEK293T-DEAF1 and HEK293T cell lines were extracted and detected using Co-IP and SDS-PAGE with EZview(TM) Red ANTI-FLAG M2 affinity gel. Bands, which were obviously enriched and did not exist in the control lanes, were analyzed with MS.


RESULTS
DEAF1 was located in the nucleus under the fluorescence microscope. Reverse transcription PCR and qRT-PCR showed that the mRNA level of DEAF1 in DEAF1-expressed HEK293T cells was higher than that in control cell line. Western blotting revealed the expression of mDEAF1-FLAG fusion protein in the transfected cells. HEK293T-DEAF1 cells which expressed DEAF1 highly and stably were obtained successfully. Some candidate proteins which might interact with DEAF1 were screened by Co-IP and MS, such as cytokines and enzymes required for pre-mRNA processing, postinitiation RNA polymerase, and other transcription factors involved in peptide bond breaking and spatial structure formatting, and so on.


CONCLUSIONS
The study obtained HEK293T-DEAF1 cells which expressed DEAF1 highly and stably as well as a large set of candidate proteins which might interact with DEAF1 using Co-IP and MS.
School of Medicine, China Three Gorges University, Yichang 443002; Department of Cardiology, Jingmen Second People's Hospital, Jingmen 448000, China.
Xianfeng
X
Wu
School of Medicine, China Three Gorges University, Yichang 443002, China.
Deqiao
D
Sheng
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D051379:Mice; D008856:Microscopy, Fluorescence; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors
D002352:Carrier Proteins; C116013:DEAF1 protein, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins