Figure 5.

Knockdown of Drob-1 enhances the accumulation of ubiquitinated proteins. (A) Fly heads from each line of the indicated genotype (1 day after eclosion) were subjected to immunoblotting with an anti-ubiquitin antibody and anti-β-tubulin antibody. (B) S2 cells were cultured with either EGFP dsRNA or drob-1 dsRNA for 48 h, and incubated with or without lactacystin (1, 5, and 20 μM) for an additional 24 h as described in Materials and methods. The cell lysate was subjected to Western analysis using the anti-ubiquitin and anti-β-tubulin antibodies. (C, D) S2 cells were transfected with EGFP, drob-1, or buffy dsRNA for 48 h, and left untreated or treated with lactacystin, a proteasome inhibitor (1, 5, 20, and 50 μM) (B), or tunicamycin, an inhibitor of N-glycosylation that induces the rapid unfolded protein response (UPR) (0.5, 2, 10, and 50 μg/ml) (C), for 24 h. Cell viability was determined by cell death assay as described in Materials and methods. Mean±s.e.m., n=3, ^*P<0.05, ^**P<0.005, and ^***P<0.0005 for cells with drob-1 dsRNA, ^†P<0.05, ^††P<0.005, and ^†††P<0.0005 for cells with buffy dsRNA as compared with control cells (with EGFP dsRNA) by paired Student's t-test. (E) Cells were cultured with or without 10 μM oligomycin, an inhibitor of mitochondrial ATP synthase, for 1 h, or with EGFP dsRNA, lacZ dsRNA, ced-9 dsRNA, drob-1 dsRNA, or buffy dsRNA for 72 h. Cellular ATP content was measured as described in Materials and methods. Each value shows the mean±s.e.m. of four (n=4) independent experiments. ^*P<0.05, ^**P<0.01, and ^***P<0.005 by paired Student's t-test.