Fig. 5.

The L69,77A NS1 protein binds to the p85β regulatory subunit of PI3K but L69,77A NS1 virus infection does not activate the PI3K/Akt pathway. MDCK cells (a) and Vero cells (b) were either mock infected, infected with the WT NS1 virus, or the L69,77A NS1 virus at an m.o.i. of 5. The p-Akt, total Akt, viral NS1 and viral NP proteins from whole-cell lysates at 5, 9, 12 and 24 h after infection were detected by Western blot. (c) 293T cells were transfected with plasmids expressing 3× FLAG-tagged WT NS1, L69,77A NS1 or p85β-binding deficient Y89F NS1 proteins. The pCMV10-3× FLAG vector without inserts was also used as a control. Cells were also co-transfected with pCMV6-XL6-p85β plasmid expressing p85β. At 24 h after transfection, 3× FLAG-NS1 and associate proteins were immunoprecipitated with anti-FLAG M2 affinity beads. FLAG-tagged NS1 protein and p85β were detected by Western blot.