DJ-1 interacting proteins analyzed by tandem affinity purification mass spectrometry
10.3760/cma.j.cn431274-20200803-01081
- VernacularTitle:采用串联亲和纯化质谱分析DJ-1的相互作用蛋白
- Author:
Wangli WEI
1
;
Tan TAN
;
Yajuan CUI
;
Can′e TANG
Author Information
1. 湖南省郴州市第一人民医院检验医学中心 423000
- Keywords:
HTB-182 cells;
Genes, DJ-1;
Interacting proteins
- From:
Journal of Chinese Physician
2021;23(10):1472-1476
- CountryChina
- Language:Chinese
-
Abstract:
Objective:RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma HTB-182 cells, then, tandem affinity purification mass spectrometry (TAP-MS) was performed to screen the interacting proteins of DJ-1 in lung cancer cell line of HTB-182.Methods:The siRNA lentivirus vector targeting DJ-1 gene was constructed to infect HTB-182 cells (DJ-1 siRNA group), and the lentivirus vector control group (control siRNA group) and blank control group were established. The expression level of DJ-1 protein was detected by Western blot, and the endogenous DJ-1 protein silenced si-DJ-1-HTB-182 cells were established. The specific primers of DJ-1 were designed, and the DJ-1 expression plasmid pNTAP-DJ-1 with streptomycin binding peptide label (SBP) and calmodulin binding peptide label (CBP) was constructed. The cell line DJ-1 siRNA HTB-182 was stably transfected with liposome, and the positive clones were screened by G418. The positive clones were verified by Western blot, and the interacting proteins of DJ-1 were found by TAP-MS.Results:The protein expression of DJ-1 in DJ-1 siRNA interference group was significantly lower than that in empty plasmid group and blank control group ( P<0.05); HTB182 cell line stably expressing pNTAP-DJ-1 plasmid was successfully constructed; Three proteins interacting with DJ-1 were screened by TAP-MS: cytokeratin 1 (keratin 1), cytokeratin 10 (keratin 10) and NADPH oxidase activating protein P47 (P47 Px). Conclusions:Keratin 1, Keratin l0 and P47 Px protein may be DJ-1 interactions protein.
