Plasmids containing RNF125 and MCM2-7 were purchased from Shanghai Cell Researcher Biotech Co., Ltd. and inserted into empty vectors, which were purchased from Cell Researcher Biotech Co., Ltd.), such as pCDH, pCDNA3.0-Myc, pET22b or pGEX4T-1. Briefly, pCDH-MCM2/MCM3/MCM6/MCM7/RNF125, pCDNA3.0-RNF126-Myc, pET22b-MCM2/MCM3/MCM6/MCM7/RNF125/UBA1/UBCH5A/catalytic core of human ubiquitin specific peptidase 2 (Usp2cc) and pGEX4T-1-MCM6/RNF125 were generated. Mutations were generated in plasmids containing RNF125 or MCM6 by site-directed mutagenesis as previously described (20 (link)). Short hairpin RNAs (shRNAs) inserted into the Plko.1 vector targeting RNF125 and MCM2, MCM3, MCM6 and MCM7 were also purchased from Shanghai Cell Researcher Biotech Co., Ltd., and the specific sequences of these shRNAs are shown in Table I .
Protocol detail
Plasmid Cloning and Mutagenesis for RNF125 and MCM2-7
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Corresponding Organization :
Other organizations : First Affiliated Hospital of Anhui Medical University, Anhui Medical University, The First Affiliated Hospital, Sun Yat-sen University, Zhongshan Hospital, Fudan University, Sun Yat-sen University, Qingdao University, Affiliated Hospital of Qingdao University, Lu'an First People's Hospital
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Variable analysis
independent variables
- RNF125
- MCM2-7
- RNF126
- UBCH5A
- Catalytic core of human ubiquitin specific peptidase 2 (Usp2cc)
- Not explicitly mentioned
- Empty vectors (pCDH, pCDNA3.0-Myc, pET22b, pGEX4T-1)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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