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Antiserum Preparation and Specific Detection of Banana RING-e3 ubiquitin-protein ligase

American Journal of Plant Sciences 2022年第3期13卷 371-379页

作者： Naitong Yu Qin Zhou Hongwei Yuan Shuli Xian Jianhua Wang Yi Yang Zhixin Liu Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences Haikou China Hainan Academy of Tropical Agricultural Resource Haikou China Hainan Key Laboratory of Tropical Microbe Resources Haikou China environment and Plant Protection Institute Chinese Academy of Tropical Agricultural Sciences Haikou China

According to the data of banana transcriptome sequencing, an e3 ubiquitin-protein ligase gene was cloned by RT-PCR method using the cDNA sample of banana leaves. The complete ORF of e3 ubiquitin-protein ligase is 681 bp long and its encoded protein showed 100% sequence identity to homologue RING-H2 finger protein (XP_009407047.1) of Musa_acuminata. Bioinformatic analysis indicated that e3 ubiquitin-protein ligase contains the Ring finger domain in C terminus and eight cross-brace motifs are found in the domain. The target gene was digested by ecoR V and ecoR I, and was inserted into prokaryotic expression vector peT-32a of the same digestions to obtain the plasmid peT32a-e3 ubiquitin-protein ligase. The recombinant plasmid was introduced into escherichia coli strain BL21 (De3), and induced at 25°C with 0.4 mmol/L IPTG for 6 hours. The soluble fusion protein was expressed and high purity fusion protein was obtained by Ni2+-NTA agarose affinity chromatography purification. The fusion protein was injected into mice 3 times to prepare the antiserum. Western blot analysis showed a specific protein band was detected in total protein sample of banana leaves, but not for the samples of wild-type Nicotiana benthamiana (N.B.) and wild-type Arabidopsis thaliana (A.T.), implying the antiserum was specific to banana e3 ubiquitin-protein ligase.

关键词： Banana e3 ubiquitin-protein ligase Antiserum Preparation Polyclonal Antibody

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Integrative proteomics reveals the role of e3 ubiquitin ligase SYVN1 in hepatocellular carcinoma metastasis

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Cancer Communications 2021年第10期41卷 1007-1023页

作者： Feiyang Ji Menghao Zhou Zeyu Sun Zhengyi Jiang Huihui Zhu Zhongyang Xie Xiaoxi Ouyang Lingjian Zhang Lanjuan Li State Key Laboratory for Diagnosis and Treatment of Infectious Diseases The First Affiliated HospitalSchool of MedicineZhejiang UniversityHangzhouZhejiang 310003P.R.China Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases HangzhouZhejiang 310003P.R.China Department of Hepatobiliary and Pancreatic Surgery The First Affiliated HospitalSchool of MedicineZhejiang UniversityHangzhouZhejiang 310003P.R.China

Background:Tumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma(HCC),but the relationship between ubiquitination and metastasis need to be studiedmore *** analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC ***:The protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by *** with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict e3 ubiquitin ligases associated with tumor *** topological network of protein substrates and corresponding e3 ubiquitin ligaseswas constructed to identify the key e3 ubiquitin ***,the growth,migration and invasion ability of LM3 and HUH7 hepatoma cell lines with andwithout SYVN1 expression interferencewere measured by cell proliferation assay,subcutaneous tumor assay,umphal vein endothelium tube formation assay,transwell migration and invasion ***,the interacting proteins of SYVN1 were screened and verified by protein interaction omics,immunofluorescence,and *** levels of related protein substrates in LM3 and HUH7 cells were compared in negative control,SYVN1 knockdown,and SYVN1 overexpression ***:In this study,our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated *** discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression ofHCC *** addition,proteomic and ubiquitinomic analyses revealed that high expression of e3 ubiquitin-protein ligase SYVN1 is related with tumor ***,we found that SYVN1 interacted with heat shock protein 90(HSP90)and impacted the ubiquitination of eukaryotic elongation factor 2 kinase(eeF2K).Conclusions:The ubiquitination profiles of HCC with and without vascular inva

关键词： cancer e3 ubiquitin-protein ligase eukaryotic elongation factor 2 kinase heat shock protein 90 hepatocellular carcinoma liver metastasis proteomics synoviolin ubiquitin

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