TTP and PARP-14 interact with a conserved ARE in TF mRNA 3′UTR. (A) ribonucleprotein complexes were immunoprecipitated by anti-TTP (T), anti–PARP-14 (P) or IgG negative control (C) from lysates of LPS-treated (1 µg/mL for 2 hours) BMDM and shown by RT-PCR to contain TF mRNA when derived from WT cells, but not when derived from Parp14^−/− or Ttp^−/− cells. HPRT provides a negative control mRNA for nonspecific pull-down. This figure shows representative gels from 3 independent experiments. (B) Western blot analysis of proteins isolated with streptavidin-coated magnetic beads from lysates of LPS-treated macrophages (1 µg/mL for 2 hours) incubated with biotinylated sense (+) or anti-sense (-) murine TF 3′UTR truncations. TTP and PARP-14 only associated with the sense full-length construct containing the ARE4 segment. In this and other figures, tubulin served as a negative marker for nonspecific protein binding to beads. (C) Similar western blots of proteins pulled down using WT TF 3′ UTR or mutant TF 3′ UTR with guanine substitutions in the conserved 17-nucleototide AREs in the critical ARE4 segment, containing a 15-nucleotide palindrome harboring 2 overlapping UUAUUUAAU nonamers (one 3′ to 5′ and the other 5′ to 3′: shown with dashed lines on ARE_WT). Intra-ARE mutation affecting both nonamers (ARE_Mut-4) abolished TTP and PARP-14 binding. (D) Nonbiotinylated TF 3′UTR transcript (ARE_WT) inhibited TTP and PARP-14 binding to biotinylated TF mRNA 3′UTR, whereas the nonbiotinylated mutant TF 3′UTR transcript (ARE_Mut-4) did not.