Fig. 3. NOP53 is involved in dephosphorylation of eIF2α for HSV-1/F replication priority.

a HeLa cells transfected with control (lanes 2, 4, 6, 8) or Flag-tagged N4 (lanes 3, 5, 7, 9) were mock-infected (lanes 1, 6, 7) or either exposed to HSV-1/F (lanes 2–5) or HSV-1/Δγ34.5 (lanes 8–9) at 10 MOI for indicated times. Cell lysates were analyzed by Western blotting with specific antibodies against HSV-ICP8, p-eIF2α, eIF2α, phospho-PKR (p-PKR), PKR, Flag and actin. The change in abundance of p-eIF2α (lanes 2–5) was analysed by densitometric analysis using Image-Pro Plus Software and normalized to eIF2α (right). b HeLa cells were transfected with YFP-tagged eIF2α (lanes 2, 4) or co-transfected with Flag-tagged N4 (lanes 3, 5) for 36 h. The cells were then mock-infected (lanes 1–3) or infected with HSV-1/F at 10 MOI (lanes 4, 5) for 12 h, and cell lysates were analyzed by Western blotting with the indicated antibodies. One of two independent experiments is shown. c HeLa cells in T-25 flask were transfected with Flag-tagged N4 (lanes 2–4) or control plasmid (lanes 5-7) (5 μg each), along with YFP-tagged eIF2α (lanes 3, 6) or YFP-tagged eIF2αS51A (lanes 4, 7) (5 μg each), then mock-infected (lane 1) or infected with HSV-1/F at 10 MOI for 12 h (lanes 2–7). Cell lysates were then analyzed by Western blotting with the indicated antibodies. One of two independent experiments is shown. d HeLa cells seeded in T-25 flask were co-transfected with control plasmid (10, 5, 0 μg) and plasmid to express GFP-tagged NOP53 (0, 5, 10 μg) for 36 h. Cells were then treated with DMSO (lanes 1–4) or 10 nM of OA (lanes 5–7) for 9 h. Electrophoretically separated proteins were analyzed by Western blotting with the indicated antibodies. e HeLa cells transfected with control plasmid (lanes 2–4) or Flag-tagged N4 (lanes 5–7) were mock-infected (lane 1) or infected with HSV-1/F at 10 MOI for 12 h (lanes 2–7) in the absence or presence of OA. Cell lysates were analyzed by Western blotting with indicated antibodies