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. 2003 Jun;23(11):3798–3812. doi: 10.1128/MCB.23.11.3798-3812.2003

FIG. 7.

FIG. 7.

Effects of affinity-purified TTP and PARN on deadenylation. Deadenylation assays were performed with the fusion proteins hTTP-FLAG or hPARN-FLAG that had been isolated by affinity chromatography from 293 cells transfected with the appropriate expression plasmids; in some cases, these were mixed with extracts from 293 cells transfected with vector alone (BS+) or CMV.hTTP.tag (hTTP). (A) Effects of affinity-purified hTTP-FLAG (T) or hPARN-FLAG (P) alone or together (TP) on the deadenylation of probe ARE-A50 in the absence (lanes 1 to 3) or presence of extracts from 293 cells transfected with vector alone (BS+) (lanes 4 to 8), either on ice (no legend) or after 60 min at 37°C (+). Lanes 9 and 10 show deadenylation of the probe in extracts from cells transfected with CMV.hTTP.tag (hTTP). The arrow indicates the migration positions of the deadenylated product of probe ARE-A50 (lanes 3, 6, 8, and 10) and the ARE probe (lanes 11 to 14). The position of probe V migration is shown in lane 15. (B) Similar extracts prepared from 293 cells were either untreated (C), extracted with phenol-chloroform (E), or boiled (B), after which they were incubated with probe ARE-A50 (lanes 1 to 4) either in the presence of FLAG peptide (F; lanes 5 to 8) or of affinity-purified hTTP-FLAG (T; lanes 9 to 12). The effects of 293 cell extracts from cells expressing transfected CMV.hTTP.tag (hTTP) on probes ARE-A50 (lanes 13 and 14) or ARE (lanes 15 and 16) are also shown. The arrow indicates the migration positions of the deadenylated product of probe ARE-A50 (lanes 10 and 14) and the ARE probe (lanes 15 and 16). The position of probe V migration is shown in lane 17.