Figure 3.

Effect of additional fusion domains. Dox time-course experiments were performed in Tet-off HeLa cells as described in Figure 2. (A) TRE-EGFP reporter constructs containing the P1 protein DE plus an RNA DE consisting of either 1 (open square), 2 (closed diamond) or 4 (open circle) copies of the nonamer, showing a progressive reduction in effective reporter half-life with increasing copy number. (B and C) The dd-Rn (B) and dd-Luc (C) constructs (open square) were fused in frame to either a puromycin-resistance gene (puro; open triangle) or a non-cleavable ubiquitin (Ub) N-terminal moiety (closed square) or both (closed triangle). The Ub moiety caused a marked reduction in effective half-life in all cases. Results are representative of at least three similar experiments (A) or are expressed as mean ± SEM (n = 3) of a single experiment (B and C).