FIGURE 5.

CUG-BP is required for deadenylation of TNFα and c-fos RNAs. (A) Extracts were depleted using anti-CUG-BP antibodies or normal serum, and CUG-BP levels were determined by Western blotting. The extent of depletion was confirmed by comparison with untreated extract (untreated lane). The band above the CUG-BP band is from immunoglobulin. The same extracts were assayed for levels of PARN before and after CUG-BP depletion by Western blotting using anti-PARN antibodies (middle panel). Blots were reprobed with anti-PMScl75 antibodies as a loading control (lower panel). (B) Extracts were either untreated, depleted for CUG-BP (α-CUG-BP), or mock-depleted (normal serum) and used in deadenylation assays over the time course indicated. Where indicated, 30 ng of recombinant CUG-BP (α-CUG-BP + rCUG-BP) was added back to the extract prior to the assay. Deadenylation for TNFα (upper panel) and c-fos (lower panel) RNAs was assessed over the time course indicated. The migration positions of the fully adenylated and deadenylated RNAs are shown. (C) Extracts were treated as in B and deadenylation of the Gem-A60 control RNA was assessed following incubation in the extracts for 40 min. The migration positions of the fully adenylated and deadenylated RNAs are shown.