Fig. 7. NOP53 is essential for γ34.5 recruitment of PP1α.

a Schematics of Flag-tagged γ34.5 mutant constructs. b HEK293T cells in T-25 flask were transfected with plasmid to express GFP-tagged N4 or co-transfected with plasmids to express GFP-tagged N4 and Flag-tagged γ34.5 for 36 h (5 μg each). Cell lysates were immuno-precipitated with mouse anti-GFP or mouse IgG antibody and subjected to Western blotting with specific antibodies to detect Flag or GFP. Whole cell lysates (WCL) were also examined to confirm the expression of proteins from the transfected plasmids. c HEK293T cells were co-transfected with plasmids to express GFP-tagged N4 and Flag-tagged γ34.5, γ34.5 variants indicated in a. Cell lysates were immuno-precipitated with mouse anti-Flag or mouse IgG antibody and subjected to Western blotting with specific antibodies to detect GFP or Flag. d HeLa cells in T-25 flask were transfected with control shRNA (lanes 1, 3) or NOP53-specific shRNA (lanes 2, 4) for 24 h, followed by transfection of Flag-tagged γ34.5 for 24 h, then infected with HSV-1/F at 10 MOI for 12 h. Cell lysates were immuno-precipitated with anti-Flag or mouse IgG antibody and subjected to Western blotting with specific antibodies to detect eIF2α, PP1α, or Flag. e HeLa cells were transfected with control shRNA or NOP53-specific shRNA followed by transfection of Flag-tagged γ34.5 (0, 5, 10 μg), then infected with HSV-1/Δγ34.5 at 0.1 MOI. Cell lysates prepared 36 h.p.i. were then analyzed by Western blotting using antibodies directed against HSV-ICP8, Flag, NOP53, and actin. f Schematic illustration of the mechanism