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. 2007 Dec 1;21(23):3135–3148. doi: 10.1101/gad.1597707

Figure 2.

Figure 2.

Comparison of deadenylation rates in HeLa cells. (A) T-Rex-HeLa cells were transfected with pFlag-CMV5/TO-G1 reporter and either pFlag-CMV5 (lanes 1–5), pFlag-hPan2 (lanes 6–10), or pFlag-hPan2 D1085A (lanes 11–15). One day later, β-globin mRNA was induced by tetracycline for 2.5 h, and then cells were harvested at the specified time after β-globin mRNA transcription shutoff. (Left) β-Globin mRNA was detected by Northern blot analysis. (Lane 16) To determine the overall poly(A) tail length and the deadenylation endpoint (arrow) of β-globin mRNA, β-globin mRNAs at the steady-state level (24-h induction) are shown. (Right) The average length of poly(A) tails at each time point was measured. (B) As in A, except that cells were transfected with pHA-CMV5 (lanes 1–5), pHA-hCaf1 (lanes 6–10), or pHA-hCaf1 D161A (lanes 11–15), and transcriptional pulse-chase analysis was performed. (C) As in A, except that cells were transfected with pCMV-Myc (lanes 1–5) or pCMV-Myc-eRF3 N (lanes 6–10), and transcriptional pulse-chase analysis was performed. (D–F) Total cell lysates were analyzed by immunoblotting using anti-Flag (D, top), anti-HA (E, top), anti-Myc (F, top), or anti-GAPDH (D–F, bottom).