Fig. 3

Caveola/raft-dependent endocytosis is required for PSV infection. a Cholera toxin B (CTB) uptake was blocked by nystatin or MβCD. PK-15 cells were mock treated or pretreated with nystatin or MβCD at indicated concentrations for 1 h, and then 10 μg/ml Alexa Fluor 594 conjugated-CTB was added and incubated at 37 °C for 60 min before observed using confocal microscopy. b-c Nystatin and MβCD reduced PSV infection. IPEC-J2 cells were pretreated with increasing concentrations of nystatin or MβCD for 1 h at 37 °C, and then cells were infected with PSV (MOI = 0.5). At 24 hpi, cells were lysed and virus titers were determined by TCID₅₀ or viral VP1 expression levels were detected by western blot. The horizontal line shows the subtoxic concentrations of nystatin or MβCD on IPEC-J2 cells. d The effect of siRNA against caveolin-1 was determined by RT-qPCR. e-f Cells were transfected with siRNA against negative control (siNC), siRNA against caveolin-1 (siCav), or cells were transfected with EGFP, EGFP-tagged wild-type caveolin-1 (Cav-WT), caveolin 1 dominant negative mutant (Y14F) (Cav-DN). At 24 h after transfection, cells were infected with PSV, and virus VP1 protein levels were detected by western blot. Data are presented as means ± SD. The error bars indicate the standard deviations of triplicate samples from one of two independent experiments. Bars,30 μm. ^*P < 0.05; ^**P < 0.01