Figure 4.

HDM2 destabilization is regulated by PI 3-kinase related kinases. (A) Half-life of HDM2 ATM phosphorylation site mutants. U2OS cells were transfected with 2 μg of HDM2 (S395A, S395D, or wt) or pcDNA3 and 125 ng GFP. Transfected cells were treated with 640 ng/ml NCS or PI for 4 h, then CHX for the indicated times. Different exposures of each transfection are shown to facilitate comparison of HDM2 stability. (B) Quantitation of HDM2 destabilization in (A) and Figure 2C. HDM2 band intensity was normalized to GFP, then normalized to the t=0 controls. (C) Half-life of HDM2 in wortmannin-treated cells. Normal human fibroblasts (WS1) were left untreated or were treated with 50 μM wortmannin for 30 min, 50 ng/ml NCS for 2 h, or wortmannin, then NCS. Carrier (t=0) or CHX was added for the indicated times. This figure is representative of three experiments. (D) Quantitation of HDM2 destabilization in (C). HDM2 band intensity was normalized to actin, then normalized to the t=0 controls. Each decrease of one unit of log₂ (band intensity) is equivalent to one half-life.