General procedures for virus binding assay ELISA Cells were seeded in 96 microtiter plate and cultured with DMEM containing 10% FBS at 37°C for 72 hours. EV71 MP4 (M.O.I = 100) or EV71 GFP were added this website into the treated or untreated cells and incubated at 4°C for 3 hours. The reactions were mixed gently every 30 minutes. After wash, the cells were fixed with 4% paraformaldehyde and incubated with anti-EV71 antibody 1 G3 at room temperature for 2 hours. Alkaline phosphatase conjugated anti-mouse
IgG (Sigma) was added and incubated at room temperature for 2 hours. After wash, substrate (p-nitrophenyl phosphate) solution was added and incubated at room temperature for 30 minutes. The reactions were quenched by adding NaOH (3.0 N) and measured the absorbance at 405 nm by EnVisonTM 2103 Multilabel reader (PerkinElmer). Flow cytometry Treated and untreated cells (4 × 105/assay) harvested
from culture plate were washed with PBS once and incubated with EV71 MP4 (M.O.I = 100) at 4°C for 3 hours. After wash, the cells were fixed with 4% paraformaldehyde and incubated with anti-EV71 antibody 1 G3 at room temperature VX 809 for 2 hours. Alexa 488 conjugated anti-mouse IgG (Invitrogen) was added into the reaction and incubated at 4°C for 1 hour. The histograms of bound viruses were analyzed by FACSCalibur flow cytometer (BD Biosciences). Real-time PCR Cells were seeded in 6 well plate (2.5 × 105/ well) and cultured with DMEM containing 10% FBS at 37°C for 72 hours. Treated and untreated cells were incubated with EV71 MP4 or 4643 (M.O.I = 10) at 4°C for 1 hour. The total RNA was extracted by RNeasy protect bacteria mini kit (QIAGEN) and the copy number of viral RNA was measured by using LightCycler RNA Master HybProbe kit (Roche). The copy number of viral RNA was calculated using a standard curve. The replication of EV71 was also
measured by real-time PCR. Treated and untreated cells were incubated with EV71 MP4 or 4643 (M.O.I = 1) at 4°C for 1 hour. After the unbounded virus was removed, culture medium was added into the well and incubated at 37°C for 24 hours. The total RNA was measured triclocarban as described above. EV71-GFP infection assay RD cells were seeded in 96 well plate (1 × 104/ well) and cultured with DMEM containing 10 % FBS at 37°C for 72 hours. Treated and untreated cells were incubated with EV71-GFP (M.O.I = 15) at 37°C for 1 hour. After the unbounded virus was removed, culture medium was added was added into the well and incubated at 37°C for 48 hours. The cell number, CPE, and fluorescence intensity were observed by fluorescence microscope at 0, 24 and 48 hours. General procedures for inhibition assays All of the inhibition assays were performed by treating cells with inhibitors, enzyme, or lectins before EV71 infection. Virus was incubated with cells at 4°C for 3 hours in binding assay, and worked at 37°C for 3 hours in virus infection assay.