FIGURE 2.
The c-fos and TNFα RNAs cross-link specifically to CUG-BP in vitro. (A) The indicated radiolabeled substrate RNA was incubated in extracts and irradiated with UV light to cross-link proteins to the RNA (input). A portion of the cross-linked proteins were subjected to immunoprecipitation (IP) with anti-CUG-BP antibody (C), or normal serum (N). In a separate experiment (ID), anti-CUG-BP antibody (C), or normal serum (N), were used to immunodeplete the extracts following cross-linking and the supernatants were analyzed. The products were separated by SDS-PAGE. The position of the cross-linked CUG-BP is indicated. (B) UV cross-linking to the c-fos RNA substrate was performed in the presence of increasing femtomolar amounts of EDEN RNA competitor or a control RNA (EDENmut), in which the CUG-BP binding sites had been mutated. The CUG-BP band is indicated. (C) UV cross-linking to the c-fos RNA substrate was performed in the presence of increasing picomolar amounts of TNFα ARE competitor or a control RNA in which the AUUUA pentamers had been mutated. The CUG-BP band is indicated, and molecular weight markers are shown.