4E2RCat

Cell experiment:

L132 cells are treated with 12.5 μM 4E2RCat for the indicated times and are processed for annexin V/propidium iodide staining. To this end, cell medium is collected. Cells are ished with 1 mL PBS, which is collected as well, and trypsinized in 200 μL 0.05% trypsin-EDTA. Cells are pooled with previously collected supernatants and spun for 2 min at 2,000 rpm and 4°C. The cell pellet is ished with 2 mL cold PBS, followed by another spin. After the second spin, the cell pellet is resuspended in 100 μL annexin V binding buffer and propidium iodide added to a final concentration of 5 μg/mL. After the addition of 5 μL annexin V-fluorescein isothiocyanate, samples are incubated for 15 min in the dark at room temperature and diluted. Fluorescence-activated cell sorter (FACS) analyses are performed using a FACScan instrument[1].

References:

[1]. Cencic R, et al. Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication. J Virol. 2011 Jul;85(13):6381-9.