Vps28

Total protein concentrations in cell lysates prepared from FMDV-infected and uninfected cells harvested at 6 h p.i. were measured, and equivalent amounts of cell lysates from two independent biological replicates were denatured by heating at 100°C for 10 min in 5×sample loading buffer and separated using 10% SDS-PAGE. Proteins were electrotransferred to 0.45 μm nitrocellulose membranes (Bio-Rad), blocked with 5% nonfat milk in TBS containing 0.05% Tween-20 (TBST) overnight at 4°C. The membranes were then incubated for 1 h at room temperature with specific antibodies against DARS (Santa Cruz Biotechnology, Inc., USA), LYPLA1 (Abcam, UK), SEC62 (Sigma-Aldrich, USA), EIF2AK2 (Abcam, UK), EVI5 (Sigma-Aldrich, USA),VPS28 (Sigma-Aldrich, USA), Tublin (Santa Cruz Biotechnology, Inc., USA)or β-actin (Santa Cruz Biotechnology, Inc., USA). Incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma) was performed at room temperature for 1 h, followed by washing with TBST three times. The protein bands were visualized using Amersham ECL Plus Western Blot Detection Reagents (GE Healthcare).

Coverslip-adhered BHK-21 cell monolayers were infected with FMDV at an MOI of 1. The plates were inoculated at 37°C over 1 h followed by replacement with fresh growth media, and were incubated at 37°C for another 5 h. The cells were fixed with 4% PFA and treated with 0.01% Triton X-100, with the following antibodies applied: DARS (Santa Cruz Biotechnology, Inc., USA), LYPLA1 (Abcam, UK), SEC62 (Sigma-Aldrich, USA), EIF2AK2 (Abcam, UK), EVI5 (Sigma-Aldrich, USA),VPS28 (Sigma-Aldrich, USA), or β-actin (Santa Cruz Biotechnology, Inc., USA). FMDV proteins were detected by pig anti-FMDV Asia 1 VLPs serum (primary antibody, 1:100) recognized by a TRITC-conjugated secondary antibody (Sigma). DAPI was used as counterstain to allow visualization of cell nuclei. Immunofluorescence confocal microscopy images were captured on an LSM510 META microscope (Carl Zeiss, Ltd.).