Overexpression of hSmg5/7a reduces the level of Upf1 phosphorylation. HEK293T cells were transiently transfected with pCI-neo-Flag-hUPF1 and either pCI-neo-Flag-hSMG5/7a or, as a control, pCI-neo. Total-cell lysates were purified by using anti-Flag antibody and incubated with or without λ-phosphatase (λ-PPase). (A) A fraction of each lysate prior to immunopurification was analyzed by Western blotting and anti-Flag antibody in order to quantitate the expression of Flag-hSmg5/7a and Flag-hUpf1 in each sample. (B) Comparable amounts of immunopurified Flag-hUpf1 were then subjected to two-dimensional gel electrophoresis under conditions that precluded detection of Flag-hSmg5/7a, and Flag-hUpf1 was localized by blotting with anti-Flag antibody.
