hSmg5/7a localizes primarily to the cytoplasmic fraction of HEK293T cells. Flag-hSMG5/7a was transiently expressed in HEK293T cells. Subsequently, RNA was purified from total-cell lysates, and protein was purified from nuclear and cytoplasmic fractions. (A) RT-PCR was used to determine the level at which Flag-hSmg5/7a was expressed relative to endogenous Smg5/7a. Considering a transfection efficiency of 90% (data not shown), the combined levels of endogenous and exogenous hSMG5/7a mRNA in transfected cells was determined to be only 2.6-fold of the level of endogenous hSMG5/7a mRNA in untransfected cells. (B) Western blotting using antibodies against PLC-γ, Flag (to detect Flag-hSmg5/7a), eRF1, eRF3, Upf1, and Upf2 was used to determine the cellular distribution of the various proteins.
