Fig. 6.

ERK1/2 knockdown suppresses PEDV infection. Vero cells were transfected with 100 nM ERK-specific siRNA or control siRNA using Lipofectamine 2000 followed by PEDV infection at 24 h post transfection. (A) At 24 hpi, cellular lysates were prepared and subjected to immunoblotting using an antibody against the ERK protein. The blot was also reacted with anti-β-actin antibody to verify equal protein leading. ERK protein expression was quantitatively analyzed by densitometry and presented in terms of the relative density value to the β-actin gene, and fold changes in the total ERK:β-actin ratio are plotted. (B) PEDV-specific CPEs were observed daily and were photographed at 24 hpi using an inverted microscope at a magnification of 200× (first panels). For immunostaining, infected cells were fixed at 24 hpi and stained with anti-PEDV N antibody followed by Alexa green-conjugated goat anti-mouse secondary antibody (second panels). The cells were then counterstained with DAPI (third panels) and examined using a fluorescent microscope at 200× magnification. Viral production was assessed exactly as described in the legend of Fig. 4B. (C) The virus supernatants were collected at 24 hpi and viral titers were determined. Values shown are representative of the mean of three independent experiments and error bars represent standard deviations. †, P<0.001.