Flag-hSmg5/7a associates with PP2Ac, Upf1, Upf2, Upf3X, and hSmg1, but not with eRF1 and eRF3. (A) Flag-hSmg5/7a and, when specified, HA-hSmg1 were transiently expressed in Cos cells, and total-cell lysate was purified by using anti-Flag antibody, which purified 26% (top) or 3% (bottom) of cellular Flag-hSmg5/7a, or anti-HA or mouse IgG, each of which controlled for nonspecific immunopurification. (B) Alternatively, lysate was purified by using microcystin (MC)-Sepharose, which purified 50% of cellular PP2Ac, or unconjugated Sepharose, which controlled for nonspecific binding to Sepharose alone. Purified material was then analyzed by Western blotting with the specified antibodies, which consisted of those against PP2Ac, Upf1, Upf2, Upf3/3X, eRF3, eRF1, Flag (to detect Flag-hSmg5/7a), and HA (to detect HA-hSmg1).
