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Fig. 6. Wt E4-ORF1, but not mutant IIB or IIIA E4-ORF1, activates the PI 3-kinase effector PKB in MDCK cells. (A) PKB activation by E4-ORF1 in MDCK cells. MDCK cells on 6 cm dishes were lipofected with pGW1-HA-PKB (1.0 µg) in combination with pGW1-wt-E4-ORF1, -IIB-E4-ORF1 or -IIIA-E4-ORF1 (50 ng). Extracts (120 µg protein) of RIPA buffer-lysed, serum-starved cells were immunoblotted with (P)Ser473PKB or HA antibodies. (B) Wt and mutant E4-ORF1 proteins are expressed at comparable levels in MDCK cells. MDCK cells on 6 cm dishes were lipofected with pGW1-HA-PKB (0.5 µg) in combination with pGW1-wt-E4-ORF1, -IIB-E4-ORF1 or -IIIA-E4-ORF1 (2.5 µg). Extracts (100 µg protein) of sample buffer-lysed, serum-starved cells were immunoblotted with E4-ORF1, or HA antibodies.





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