Fig. 3. Cdk5 stabilizes p53 in the presence of Hdm2. (A) H1299 cells were transiently co-transfected with a fixed amount of vectors encoding p53/Hdm2/Cdk5 (0.5 µg/1.5 µg/2 µg) and the various amounts of vector encoding p25 (0, 2 and 4 µg). Cell lysates were subjected to immunoblotting with the indicated antibodies. (B) In similar experiments as described in A, cells were transfected with a fixed amount of p53/Hdm2/p25 (0.5 µg/1.5 µg/2 µg) as indicated. For the inhibition of Cdk5, cells were transfected with Cdk5(D144N) instead of Cdk5 as indicated. (C) Mouse embryonic fibroblasts derived from double-knockout mice (p53^–/–/Mdm2^–/–) were transiently co-transfected with a fixed amount of vectors encoding p53/Cdk5 (0.5 µg/2 µg) and the various amounts of vector encoding p25 (0, 2 and 4 µg). Cell lysates were subjected to immunoblotting with the indicated antibodies. (D,E) H1299 cells were transfected with the indicated combinations of vectors encoding p53 (0.5 µg), Hdm2 (1.5 µg), Cdk5-D144N (2 µg) or p35/p25 (2 µg). 20 µM MG132 was added 36 hours after transfection for 8 hours. (i) Total p53 was immunoprecipitated with a polyclonal antibody and detected with a monoclonal antibody. The ubiquitylated form of p53 is indicated. EGFP was used as the transfection control. (ii) For detecting the association of p53 with Hdm2, cell lysates were immunoprecipitated with anti-p53 and then immunoblotted with anti-Hdm2, with 5% of the cell lysates used as input controls.