FIG. 4.
Upf2p is phosphorylated in vivo. (A) A wild-type strain (lane 1), a upf2Δ strain (lane 2), and a upf2Δ strain expressing FLAG-Upf2p (lane 3) were labeled with [32P]orthophosphate and immunoprecipitated with anti-Upf2p polyclonal antibody. Labeled proteins were detected by phosphorimaging. (B) Immunopurified FLAG-Upf2p was incubated with or without calf intestinal phosphatase (CIP) and analyzed by Western blotting with anti-Upf2p polyclonal antibody. (C) In vivo labeling experiment, performed as described for panel A, with yeast upf2Δ strains expressing the indicated upf2 constructs. The assay was performed as described for panel A, except that the cytoplasmic extracts were immunoprecipitated with anti-FLAG antibody and detected by autoradiography (upper panel) or by Western blotting with anti-FLAG antibody (lower panel). The asterisk indicates an N-terminally truncated Upf2p product.