Figure 5

CD437-dependent RNA-protein interactions are independent of nascent protein synthesis or transcription but are phosphorylation dependent. The preparation of cytoplasmic protein extracts from untreated (lanes 0 hour) or CD437-treated (lanes 2, and 4-6, 6 hours) MDA-MB-468 HBC cells, and transcription and labeling of the indicated probe as well as in vitro binding reactions were as described in the methods. The cytoplasmic extracts were prepared from HBC cells pre-treated with protein synthesis inhibitor CHX (lanes 1, 2) or transcriptional inhibitor ActD (lanes 3, 4) followed by treatments with CD437 (time and dose indicated). In addition, cytoplasmic protein extracts derived from CD437-treated HBC cells were pretreated with alkaline phosphatase (lane 6) or proteinase K (lane 5) prior to binding with the indicated probe. The approximate migration of molecular weight standards is marked on the left side, whereas the * and ** on the right side of the panel denote locations of the putative 85 kD and 55 kD complexes, respectively.