Figure 4

Multiple cell cytoplasmic proteins bind to radiolabelled p21 ^WAF1/CIP1 3'-UTR and GADD45 5'-UTR sense strand riboprobes. Transcription and labeling of the indicated probes as well as the in vitro binding reactions were as described in the Methods. The probes utilized and treatment times with CD437 (hr) are indicated above each lane in the respective panels. The cytoplasmic protein extracts were prepared from untreated (lanes marked 0 hours) or CD437-treated MDA-MB-468 (panels A-C), MCF-7 (panel C) and HL-60R (panel C) cells. Panel A, the protein extracts in lanes 4-6 and11-13 were pre-incubated with 200-fold excess of the respective unlabeled probe RNAs, while the protein extracts in lane 7 were pre-incubated with 200-fold excess of the unlabeled 3'-UTR of c-myc RNA followed by incubation with the indicated labeled probe RNAs. Probe 14.2 RNA is loaded in lane 14. Panel B, the protein extracts in lanes 3 and 6 were pre-incubated with 200-fold excess of the respective unlabeled probe RNAs, while the protein extracts in lane 7 were pre-incubated with 200-fold excess of the unlabeled 3'-UTR of c-myc RNA followed by incubation with the indicated labeled probe RNAs. Panel C, lanes 6, 12 represent the labeled probe only. 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.