FIG. 7.
Half-life of mdm2 is decreased by IE2-86: pulse-chase. H1299 cells were transfected with expression vectors encoding mdm2 and the indicated combinations of p53 and IE2-86 and pulsed with [35S]Met and then chased with unlabeled amino acids for the indicated times prior to harvesting. (A) Labeled mdm2 was detected by immunoprecipitation followed by autoradiography and the levels of other proteins were determined by immunoblotting. (B) The autoradiographed film was analyzed with New ImageQuant TL v2003.01 to detect relative levels of labeled mdm2 at the indicated chase time points. (C) The amounts of p53, mdm2, and IE2-86 were determined by immunoblotting at the indicated chase time points. “a” refers to transfection with mdm2, “b” refers to transfection with mdm2 and p53 and IE2-86, “c” refers to transfection with mdm2 and p53, and “d” refers to transfection with mdm2 and IE2-86. IP, immunoprecipitation; W, Western blotting; Ub, ubiquitin; IgG, immunoglobulin G.