Fig. 6
The initial decrease of mdm2 expression following a high dose of UV irradiation is transcriptionally regulated
SP2/0 cells were treated with a UV dose of 20 J/M2 and harvested at the indicated times. The protein levels of Mdm2 and p53 were analyzed by IP-Western (A) and the mRNA levels of mdm2, p21/WAF1, and ß-actin were examined by Northern analysis (B). (C) Northern analysis of stability of the mdm2 mRNA in SP2/0 cells treated with or without UV (20 J/M2). SP2/0 cells were first treated with actinomycin D at a concentration of 0.5 µg/ml for 15 min and then were either irradiated with 20 J/M2 UV or were left untreated. Total RNA was isolated from the cells at the indicated times. 10 µg of each total RNA was used for Northern blots probed with cDNAs of mdm2 and ß-actin. (D) The relative amounts of mdm2 mRNA levels to the un-irradiated level were plotted as a function of time. (E) Nuclear run-on assays. The experimental procedure was described in Materials and Methods. The membranes were blotted with cDNAs of mdm2, p21/WAF1, GAPDH, ß-actin, and vector pBluescript. Both light (top) and dark exposures (bottom) were taken for showing different intensities of signals on the same blots. The mdm2 signal as indicated was shown on the light exposure while the signals of p21/WAF1, GAPDH, and ß-actin only appeared on the dark exposure. (F) Effects of UV on transcription of various genes.