FIG 1.

PSaV-induced early activation of PI3K/Akt and MEK/ERK signaling pathways. (A and B) LLC-PK cells were inoculated with the PSaV Cowden strain (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA (bile acid) and then harvested at the indicated time points. The levels of PI3K, Akt, ERK, pPI3K p85 (Tyr458)/p55 (Tyr199), pAkt (Ser473), pERK (Thr202/Tyr204), and GAPDH were evaluated by Western blotting using specific antibodies against the target proteins. GAPDH was used as a loading control. (C) LLC-PK cells were mock pretreated or pretreated with wortmannin (PI3K inhibitor) or U0126 (MEK inhibitor) at the indicated doses for 1 h at 37°C and then infected with or without PSaV in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 min postinoculation (mpi). The expression levels of pAkt (Ser473), Akt, pERK (Thr202/Tyr204), ERK, and GAPDH were evaluated by Western blotting. GAPDH was used as a loading control. (D) LLC-PK cells transfected with scrambled siRNA (Scram) or siRNA against PI3K p85α or MEK were harvested at 24 and 48 h posttransfection. The downregulation of each protein by siRNA knockdown was evaluated by Western blotting using antibodies specific for each protein. GAPDH was used as a loading control. (E) LLC-PK cells transfected with or without each siRNA were incubated with PSaV (MOI of 1 FFU/cell) in the presence of 200 μM GCDCA. Cell lysates were harvested at 5 mpi. The expression levels of pAkt (Ser473), PI3K, pERK (Thr202/Tyr204), MEK, and GAPDH were determined by Western blotting. GAPDH was used as a loading control. The intensity of each target protein relative to that of GAPDH was determined by densitometric analysis and is indicated above each lane.