Fig. 1. (A) Extensive modification of NuMA in HSV-infected cells. HEp-2 cells infected with HSV-2 or HSV-1 at a MOI of 3 PFU/cell were harvested at the indicated time points and analysed by SDS-PAGE and western blotting with a NuMA polyclonal antibody. The slower migrating band is prominent at later times post infection. (B) NuMA is phosphorylated in HSV-infected cells. Uninfected HEp-2 cells (lanes 1, 2) or HEp-2 cells infected for 17 hours with HSV-2 at a MOI of 5 PFU/cell (lanes 3, 4) were harvested and dephosphorylated with phosphatase (+) or incubated without phosphatase (–). NuMA in HSV-2-infected cells was phosphorylated (lane 3) and phosphatase treatment resulted in a faster migrating band (lane 4). (C) Blockage of viral DNA synthesis by PAA treatment reduces modification of NuMA in HSV-2-infected cells. HEp-2 cells were infected with HSV-2 at a MOI of 5 PFU/cell in the absence (lane 2) or presence (lane 3) of 300 µg/ml PAA and harvested at 17 hours post infection. Mock-infected cells were harvested before infection (lane 1) and also 17 hours later (lane 4). (D) The Cdc2 consensus sites in the C-terminal region of NuMA (Thr2000, Thr2040 and Ser2072) are not modified in HSV-2-infected cells. HEp-2 cells were transfected with pFLAG-NuMAwt and pFLAG-NuMAcdc2 and 24 hours later were mock-infected (lanes 1, 3) or infected with HSV-2 at a MOI of 3 PFU/cell for 15 hours (lanes 2, 4). The cells were harvested and analysed by SDS-PAGE and western blotting with an anti-FLAG monoclonal antibody. The motility of the bands in lanes 2 and 4 are nearly identical, suggesting that NuMA was not modified at the Cdc2 sites.