FIG 3.

Dynein activation by HSV-1. (A) Serum-starved Vero cells were infected with HSV-1 (McKrae) at 4°C for 1 h at an MOI of 5 and then shifted to 37°C for 15 min, 30 min, 1 h, 2 h, and 3 h. Cells were lysed with the NP-40 lysis buffer containing a protease inhibitor cocktail and analyzed for phosphorylated dynein intermediate chain (DIC 1B S80A) by SDS-PAGE. The total dynein intermediate chain (DIC) protein was used as the loading control. (B) Vero cells were pretreated with miltefosine at 37°C. Cells were infected with HSV-1 (McKrae) at 4°C for 1 h at an MOI of 5 and then shifted to 37°C for 1 h. Cells were lysed with NP-40 lysis buffer containing a protease inhibitor cocktail and analyzed for the presence of the phosphorylated dynein intermediate chain (DIC 1B S80A) by SDS-PAGE. (C) Following infection with HSV-1 (McKrae) at 4°C for 1 h at an MOI of 5, cells were incubated at 37°C for 15 min, and then miltefosine was added and the mixture was incubated for 1 h. Cell lysates were prepared the same way for SDS-PAGE analysis. + and − indicate whether serum was added (+) or not (−). (D) Effect of miltefosine on Akt phosphorylation. Total Akt was used as loading control.