Figure 6

Multiple CD437-responsive sequence elements exist within the 3'-UTR of p21 ^WAF1/CIP1. Transcription and labeling of the indicated probes and binding reactions were as described in the Methods. The probes utilized and treatment times with CD437 (hours, hr) are indicated in the respective panels. In panel B, the probe sequence of the 12 nt RNA oligonucleotide is shown. The cytoplasmic protein extracts were prepared from untreated (lanes marked 0) or CD437-treated MDA-MB-468 (panels A, B), MCF-7 (panel B) and HL-60R (panel B) cells. The respective probes only were loaded in lanes 1, 5, 9, and 13. The protein extracts in lanes 4, 8, 12, and 16 were pre-incubated with 200-fold excess of the unlabeled RNA derived from pBKS-WAF (1540-1700) plasmid (table 2), followed by incubation with the indicated labeled probe. The approximate migration of molecular weight standards is marked on the left side of respective panel, while the * and ** on the right side of each panel denote locations of the putative 85 kD and 55 kD complexes, respectively.