Confirming direct association between APE1 and p53 protein in A549 and HeLa cells
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Confirming direct association between APE1 and p53 protein in A549 and HeLa cells
Journal of Army Medical UniversityVol. 35, Issue 21, Pages: 2314-2319(2013)
作者机构:
第三军医大学大坪医院野战外科研究所:肿瘤中心,病理科
作者简介:
基金信息:
DOI:
CLC:R394.3;R394-33;R730.23
Published:2013,
Published Online:15 November 2013,
稿件说明:
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ZHANG CHONG, LI MENGXIA, CHENG YI, et al. Confirming direct association between APE1 and p53 protein in A549 and HeLa cells. [J]. Journal of army medical university, 2013, 35(21): 2314-2319.
DOI:
ZHANG CHONG, LI MENGXIA, CHENG YI, et al. Confirming direct association between APE1 and p53 protein in A549 and HeLa cells. [J]. Journal of army medical university, 2013, 35(21): 2314-2319.DOI:
Confirming direct association between APE1 and p53 protein in A549 and HeLa cells
Objective To investigate the endogenous and exogenous interaction between apurinic/apyrimidinic endonuclease 1 (APE1) and p53 in A549 and HeLa cells. Methods Recombinant plasmid pET42a-APE1 was transformed into
E. coli
BL21(DE3)
and the expression of APE1was induced and identified with IPTG. The expressed GST-APE1 fusion protein was purified by affinity chromatography with Ni
2+
column and identified by We
stern blotting. The interaction between APE1 and p53 protein was identified by co-immunoprecipitation (Co-IP) and GST pull down assay. Immunofluorescence assay and laser scanning confocal microscopy was used to observe the subcellular localization of the 2 proteins. Results Highly expressed hAPE1 fusion protein by pET42a-hAPE1 was obtained in
E.coli
BL21(DE3) after IPTG induction. The GST-APE1 fusion protein with molecular weight of 64×10
3
by SDS-PAGE was purified by affinity chromatography with Ni
2+
column and identified by Western blotting. Through Co-IP and GST pull down assay
APE1 was confirmed to interact with p53 endogenously and exogenously. Immunofluorescence assay and laser scanning confocal microscopy indicated that the 2 proteins were located in the nuclear and cytoplasm
and then transformed from the cytoplasm to the nuclear membrane after oxidative stress. Conclusion There are endogenous and exogenous interaction between APE1 and p53 protein in A549 and HeLa cells
which might be associated with transcriptional regulation and efficiency of tumor radiotherapy and chemotherapy.
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