FIG. 2.
(A and B) Immunoblots of electrophoretically separated Us3 immunoprecipitates (upper panel) and lysates (lower panel) from Vero cells infected with HSV-1(F). Infected cells were harvested at 18 h postinfection, solubilized, and either mock treated (lanes 1) or treated with λ-PPase (lanes 2). Part of the treated cell lysates was analyzed by immunoblotting with anti-Us3 antibody (lower panels). The remainder of each lysate was immunoprecipitated with anti-Us3 (A) or anti-Us3-S147P antibody (B). The immunoprecipitates were separated in a denaturing gel and analyzed by immunoblotting with anti-Us3 antibody (upper panel). (C and D) Amount of Us3 protein immunoprecipitated with anti-Us3 (B, upper panel) and anti-Us3-S147P antibody (C, upper panel) relative to the amount of Us3 protein in the corresponding HSV-1(F)-infected cell lysates (A and B, lower panels). The data were normalized to the value for HSV-1(F)-infected cells without phosphatase treatment in panels A and B, lanes 1. (E) Immunoblots of electrophoretically separated Us3 immunoprecipitates (upper panel) and lysates (lower panel) from Vero cells infected with HSV-1(F) (lane 1) or YK515 (Us3-S147A) (lane 2). Infected Vero cells were harvested at 18 h postinfection and solubilized. Part of the cell lysates was immunoblotted with anti-Us3 polyclonal antibody (lower panel). The remainder of each lysate was immunoprecipitated with anti-Us3. The immunoprecipitates were separated in a denaturing gel and analyzed by immunoblotting with anti-Us3 antibody (upper panel). (F) Amount of Us3 protein immunoprecipitated with anti-Us3 antibody (E, upper panel) relative to the amount of Us3 protein in the corresponding HSV-1(F)- or YK515 (Us3-S147A)-infected cell lysates (E, lower panel). These data were normalized relative to the value for HSV-1(F)-infected cells in panel E, lane 1. IP, immunoprecipitation; IPs, immunoprecipitates; IB, immunoblotting; α, anti; +, present; −, absent.