A general strategy to inhibiting viral -1 frameshifting based on upstream attenuation duplex formation

Hu, Hao-Teng; Cho, Che-Pei; Lin, Ya-Hui; Chang, Kung-Yao

The 293T cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) on 10-cm dishes to 90% confluency. Cells were then transferred to 15-cm dishes, incubated to 90% confluency and then washed with ice-cold phosphatebuffered saline (PBS) and detached by trypsinization using trypsin-EDTA (0.05% Trypsin with 2 mM EDTA, Gibco). After stopping trypsinization with medium containing 10% FBS (Corning), the detached cells were centrifuged at 1000 g at 4 • C to pellet the cells. The cell pellet was washed twice with PBS and re-suspended with hypotonic buffer (20 mM HEPES (pH7.5), 100 mM potassium acetate, 1 mM Magnesium acetate, 2 mM dithiothreiol (DTT) and proteinase inhibitor cocktail (Roche)). Re-suspended cell pellets were incubated on ice for 45 min and homogenized through a 1 ml syringe using 26 G, 3/4-inch needle (34) . After centrifugation at 14 000 g for 1 min at 4 • C, the supernatant containing cell lysate was collected with the contents of protein concentration measured by Bradford assay (Biorad), and stored at −80 • C.