Hemagglutinin (HA)–tagged WT, the arginine to lysine 111/113 mutant E2F1 (KK), and the arginine to lysine 109 (R109K) constructs have been described previously (11). These were subcloned into a pTRE2-hyg expression vector (Clontech) and transfected into parental Tet-On U2OS cells (Clontech; RRID: CVCL_V335) to generate inducible, stable cell lines. These cells were selected in Dulbecco’s modified Eagle medium (DMEM) (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin/streptomycin, G418 (100 μg/ml; Santa Cruz Biotechnology), and hygromycin B (150 μg/ml; TOKU-E). For all experiments, doxycycline (1 μg/ml) was used to induce protein expression for 24 hours before harvest. E2F1 and TSN CRISPR cells were generated as per the protocol described (28) and cultured in DMEM containing 10% (v/v) FBS and penicillin/streptomycin. All cell lines were tested for mycoplasma contamination before 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.