FIG. 3.

Identification of additional GC-rich RNA binding proteins after separation from RNA. (A) The left-hand gel shows gelshift analysis of DEAE fractions (shown at the top) with the sORF probe. The positions of GCU- and sORF-specific complexes are indicated. C, unfractionated cytoplasm. −, no protein added. The right-hand gel shows gel shift and competition analyses of the 0.2 M NaCl elution fraction. Cold competitors (indicated at the top) were added to the binding reaction before the addition of probe. GCU, L (LAP), and sORF, competitors that contain GC-rich sequences (see Materials and Methods) and interact with CUGBP1 (35); GX, AU-rich oligomer with random sequence (35). free, position of free sORF probe. (B) (Top) UV cross-link analysis of DEAE fractions with a short (GCU)₈ probe. Cytoplasm (C) and elution fractions (shown at the top) were incubated with the (GCU)₈ probe, linked by UV treatment, and separated by SDS-gel electrophoresis. The positions of the CUGBP1, p60, and p150 binding proteins are shown on the right. (Bottom) Western blot of DEAE fractions with monoclonal antibodies to CUGBP1 and with antibodies to EXP42. (C) RNA-free CUGBP1 and p60 bind strongly to RNA containing long GCU repeats. Both gels show the interaction of GC-rich RNA binding proteins with RNA transcripts containing 39 GCU repeats (left) or 6, 39, and 123 GCU repeats (right). C, unfractionated cytoplasm. The position of EXP42 was determined by reprobing the membrane with antibodies to EXP42.