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. 2013 May 10;288(24):17832–17843. doi: 10.1074/jbc.M112.448977

FIGURE 3.

FIGURE 3.

Non-stop mRNA decay requires Hbs1 and Dom34. A, down-regulation of Hbs1 and Dom34 expression. HeLa cells were transfected with control luciferase siRNA (lane 1), Hbs1 siRNA-1 (Hbs1(#1); lane 2), or Dom34 siRNA-1 (Dom34(#1); lane 3). Total cell lysate was analyzed by Western blotting (WB) using the indicated antibodies. B, down-regulation of either Hbs1 or Dom34 expression inhibits fast degradation of non-stop mRNA. HeLa cells were transfected with p5×FLAG-EGFP; pT7-TR; either pFBG control (lanes 1–5, 11–15, and 21–25) or pFBG non-stop (lanes 6–10, 16–20, and 26–30); and control luciferase siRNA (lanes 1–10), Hbs1 siRNA-1 (lanes 11–20), or Dom34 siRNA-1 (lanes 21–30). The transcriptional pulse-chase analysis was performed as described in the legend to Fig. 2, except that the siRNAs were cotransfected. C, the levels of FBG mRNAs were quantitated as described in the legend to Fig. 2. D, the half-lives of FBG mRNAs were calculated. The asterisks denote the levels of statistical significance (Student's t test) between luciferase siRNA and either Hbs1 or Dom34 siRNA: *, p < 0.05; **, p < 0.01.