p3XFLAG-Snail-WT, p3XFLAG-Slug-WT, and pLKO.1-ms.p53-shRNA vectors were generated and used as described previously (20, 21). pET23a(+)-His-Snail-WT, His-Snail-R174A, p3XFLAG-Snail-R174A, FLAG-Snail-K147R/K186R, pLKO.1-hu.p53-shRNA (targeting mRNA sequence from ATG, 176 to 196), pLKO.1-Snail-shRNA1 (468 to 486), pLKO.1-Snail-shRNA2 (1515 to 1533), pCDN3.1-GST-Snail-WT-GFP, and pCDN3.1-GST-Snail-R174A-GFP vectors were generated by GenScript Biotech Inc. (Nanjing, China). HA-ubiquitin (#18712), GST-CBP-HAT (#21093), pLKO.1-TRC (#10879), psPAX2 (#12260), and pMD2.G (#12259) were purchased from Addgene. To produce pLKO.1 lentiviral particles, HEK293T cells were cotransfected with pLKO.1-shRNA, psPAX2, and pMD2.G at a ratio of 4:3:1 using Lipofectamine 2000 Reagent (Invitrogen, #11668027). Cells were fed with fresh medium 24 hours after transfection, and conditioned medium containing viral particles was harvested 48 and 72 hours after transfection. Viral particles were stored at −80°C for further use or immediately used. For lentiviral infection, target cells were incubated with a mixture of conditioned medium (containing viral particles) and culture medium at a ratio of 1:1 for 24 hours in the presence of polybrene (8 μg/ml; Sigma-Aldrich, #H9268). Cells were reinfected with viral particles for another 24 hours and harvested for further use. For adenoviral infection, cells were infected with complete medium supplemented with adeno-βGal or adeno-Cre viral particles for 24 hours, refed with fresh medium containing viral particles, and further cultured for another 24 hours. Cells were collected for further use.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.