hUpf2 is a phosphoprotein, but overexpression of hSmg5/7a does not reduce the level of hUpf2 phosphorylation. HEK293T cells were transiently transfected with pCI-neo-T7-hUPF2 and either pCI-neo-Flag-hSMG5/7a or, as a control, pCI-neo. Total-cell lysates were purified by using anti-T7 antibody and incubated with or without λ-phosphatase (λ-PPase). (A) A fraction of each lysate was analyzed by Western blotting and anti-T7 or anti-Flag antibody in order to demonstrate the presence of T7-hUpf2 and Flag-hSmg5/7a. (B) Comparable amounts of immunopurified T7-hUpf2 were then subjected to two-dimensional gel electrophoresis, and T7-hUpf2 was localized by Western blotting and anti-T7 antibody.
