Location Verification of NLS-RARα Protein in Infected NB4 Cell Line with Adenovirus Ad-NE

Wang Hui^1,2, Zhong Liang², Jiang Kailing², Zhu Xinyu², Ma Pengpeng², Yang Xiaoqun², Liu Beizhong^1,2*

¹Central Laboratory of Yongchuan Hospital, Chongqing Medical University, Chongqing 402160, China; ²Key Laboratory of Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqi

Abstract: The experiment mainly inverified the presence and location of NLS-RARα protein in NB4 cells infected with recombinant adenovirus Ad-NE. In our study， recombinant adenovirus Ad-NE was used to infect NB4 cells and then using FACS to confirm the efficiency of infection; RT-PCR and Western blot were used to identify the infection success in mRNA and protein levels; We extracted the nucleoprotein from successfully infected NB4 cells， and determined the expression of NLS-RARAα in NB4 cell nucleus by Western blot; FITC-Annexin V/DAPI double staining immunofluorescence and confocal laser scanning method were used to examine NLS-RARAα protein expression and localization in infected NB4 cells. The result showed that the infection efficiency of recombinant adenovirus Ad-NE and negative control adenovirus Ad-KZ in NB4 cells were up to 70%~80%. The results of RT-PCR and Western blot showed that NE gene and protein were successfully expressed in NB4 cells infected with recombinant adenovirus Ad-NE， and there did exist NLS-RARα expression. Using cell immunofluorescence and confocal laser scanning， we found that NLS-RARα protein was present in infected NB4 cells and mostly localized in cell nucleus. In summary， we have successfully infected NB4 cells with recombinant adenovirus Ad-NE and verified the existence of NLS-RARα protein with Western blot， immunofluorescence and confocal laser scanning methods. NLS-RARα was mainly localized in nucleus. These results provided a new way for further study in early diagnosis and monitoring of acute promyelocytic myeloid leukemia.

CSTR: 32200.14.cjcb.2014.03.0007