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Figure 2


Fig. 2. Dnr1 affects Dronc protein levels. (A) Real-time PCR measurement of dnr1 transcript levels in control S2 cells (column 1) and S2 cells incubated with Dnr1 dsRNA for 72 hours (column 2). dnr1 transcript levels were normalized to actin transcript levels in both samples. Dnr1 dsRNA decreases dnr1 transcript levels by greater than 95% within three days. (B) Western blot analysis of Dronc protein in lysates from control S2 cells (lanes 2-5) and S2 cells incubated with Dnr1 dsRNA (lanes 7-10). The cells were treated with actinomycin D and lysates harvested at the indicated periods. Molecular mass markers are shown in lanes 1 and 6, actin levels are shown as loading controls (lower panel) and the full-length (FL), Pr2 and L Dronc isoforms are indicated. (C-E) Quantification of the (C) full-length (FL), (D) Pr2 and (E) large (L) forms of Dronc in control S2 cells or S2 cells treated with Dnr1 dsRNA. Dronc protein levels are normalized relative to actin protein levels. The Dronc:actin levels in control S2 cells at 0 hours actinomycin D treatment were assigned a value of 1 in (C) and the remaining Dronc:actin values are reported relative to this value. We detected a significant increase in full-length Dronc protein levels in S2 cells incubated with Dnr1 dsRNA compared with control cells in the absence of induction. Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.03, values that differ significantly from untreated cells. We also detected elevated levels of the Pr2 and large isoforms of Dronc in cells treated with Dnr1 dsRNA (D and E, respectively). Results are the mean of three independent experiments and error bars indicate standard errors. *P<0.01, values that differ significantly from untreated cells. (F) Time course of induction of HADnr1 or HADnr1C563Y. HADnr1 and HADnr1C563Y levels are normalized to actin levels. (G) Levels of full-lenght Dronc at various times after induction of HADnr1 or HADnr1C563Y. Full-lenght Dronc levels are normalized to actin levels in both samples.