FIG. 3.

The cleaved form of 4E-BP1 does not undergo efficient insulin-stimulated phosphorylation. (A) HEK293 cells overexpressing either 4E-BP1myc/his or Δ24/BP1. In vivo radiolabeling was carried out as described in Materials and Methods and ³²P label incorporation into 4E-BP1 was quantified ([³²P]-label; the 4E-BP1myc/his serum-starved-only sample is standardized to 1). (B) HEK293 cells overexpressing either 4E-BP1myc/his or Δ24/BP1 at comparable levels were treated as indicated. The extracts were analyzed for total 4E-BP1 levels and phosphorylation of 4E-BP1 at Thr36 and 45, Ser64, and Thr69 as indicated.