Ripa lysis buffer

The anti-EV71 activity of LJ04 was also assayed using the CCK-8 method. In brief, the MA104 cells (1 × 10 4 cells/well) were plated into 96 well plates and incubated overnight. The medium was then removed. EV71 at 100 TCID 50 was added to the cells with or without LJ04 at a certain concentration obtained by dilution with RPMI-1640 containing 2% FBS. Each experimental sample was tested in triplicate wells. The cells were then incubated for another 96 hr. The cytoprotective activity of LJ04 was then determined using CCK-8 assay kits according to the manufacturer's instructions. This experiment was repeated using two concentrations (1 and 10 µg/ml). Total protein samples were obtained by extraction with radioimmunoprecipitation assay (RIPA) lysis buffer (Qiagen, Hilden, Germany) on ice for 30 min and centrifugation at 12,000g and 4°C for 10 min, and the supernatants were collected for the detection of EV71 VP1 expression by western blot analysis.

The cells were lysed with RIPA lysis buffer (Qiagen, Hilden, Germany) on ice for 30 min and centrifuged at 12,000g and 4°C for 10 min, and then the supernatants were collected. The concentrations of proteins were determined using the BCA Protein Assay Kit (Pierce, Waltham, MA). Equal concentrations of proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Whatman, Marlborough, MA). The membranes were blocked in skim milk dissolved in Tris-buffered saline and Tween 20 (TBST) buffer (5% wt/vol) for 2 hr at room temperature and then incubated with monospecific antibodies (anti-2A, anti-3C, and anti-βactin clone: BA3) or polyclonal antibody (anti-eIF4G; Absin Bioscience Inc., Shanghai, China) overnight at 4°C. Thereafter, the membranes were washed three times with TBST, incubated with the corresponding secondary antibodies (Absin Bioscience Inc., Shanghai, China), and washed again with TBST. The immunoreactive proteins were visualized by color development and imaged with a Tanon 6200 Luminescent Imager (Tanon Science & Technology Co., Ltd., Shanghai, China). This experiment was independently performed at least three times.