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Fig. 7. Dnr1-dependent destruction of Dronc is pro-domain independent. (A) Western blot analysis of lysates from control S2 cells (lanes 1 and 2), and S2 cells constitutively expressing HADnr1 or HADnr1C563Y (lanes 3 or 4, respectively). The individual lines were transfected with a plasmid that drives expression of 6xmycDronc where indicated. Lysates were probed with anti-myc (red) and anti-actin antibodies (green). Three distinct Dronc isoforms were detected corresponding to the full-length (FL), D135 and D113 isoforms. Each isoform is indicated by an arrow. (B) Serial dilutions of lysates from S2 cells, HADnr1-expressing cells and HADnr1C563Y-expressing cells transfected with a 6xmycDronc expressing plasmid were analyzed by western blot analysis. The levels of the individual Dronc isoforms were quantified and normalized to the level of a control protein (actin). Results are the mean of three independent experiments and error bars indicate standard errors. The level of each Dronc isoform is significantly reduced in HADnr1-expressing cells compared with S2 cells, whereas HADnr1C563Y expression fails to impact on Dronc protein levels. (C) Equal amounts of a plasmid that drives expression of 6xmycDroncPro was transfected into S2 cells, HADnr1-expressing cells and HADnr1C563Y-expressing cells as indicated. The myc tag was visualized by western blot analysis (upper panel) and quantified (lower panel) relative to the levels of a control protein (actin, middle panel). Overexpression of Dnr1 decreases the levels of DroncPro, whereas the C563Y variant of Dnr1 has no significant impact on DroncPro levels. Results are the mean of three independent experiments and error bars indicate standard errors.

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