Figure 1: Pellino negatively regulates Toll signalling in Drosophila.

(a) S2 cells were transfected with the expression empty or different doses of Toll^ΔLRR vectors together with Drs-luc and Renilla-luc plasmids. Thirty-six hours after transfection, cells were lysed for luciferase assays (upper panel) and immunoblotting assays (lower panel). Luciferase activity was measured and normalized based on renilla luciferase activity. Error bars represent s.d. (n=3). (b) DsRNAs targeting gfp, cactus (negative control), pelle (pll, positive control) or three different regions of pellino were treated in S2 cells for 48 h, then the empty vector or Toll^ΔLRR plasmids were transfected in S2 RNAi cells with Drs-luc and Renilla-luc plasmids. At 36 h post-transfection, cells were lysed for luciferase assays (upper panel), and immunoblotting with a Pellino-specific antibody to confirm RNAi efficiency (lower panel). Error bars represent s.d. (n=3). (c–e) S2 cells were treated with dsRNAs targeting gfp, cactus, pelle or pellino. After 48 h treatment, cells were transfected with DNA vector expressing Toll^ΔLRR. At 36 h post-transfection, total RNA was isolated for quantitative RT-PCR to determine transcriptional levels of drosomycin (c) metchnikowin (d) and defensin (e). Error bars represent s.d. (n=3). (f) S2 cells were pretreated with gfp, cactus, pelle and pellino dsRNAs for 48 h and then were transfected with vectors expressing Toll^ΔLRR. Forty-eight hours after transfection, cells were lysed for western blot assays to measure protein levels of Drosomycin and Defensin. Actin is shown as loading control. (g) S2 cells were treated with dsRNA (as indicated) for 48 h, reporter plasmids and the indicated expression vectors were transfected into dsRNA knockdown cells. After 36 h, cells were lysed for luciferase assays (upper panel), and immunoblotting assays (lower panel). Error bars represent s.d. (n=3). (h) S2 cells were transfected with vector or expression constructs as indicated. Thirty-six hours after transfection, cells were extracted for reporter assays (upper panel) and immunoblotting assays performed with the indicated antibodies (lower panel). Error bars represent s.d. (n=3). For data from (b–e, g,h), the two-tailed Student’s t-test was used to analyse statistical significance. *P<0.05, **P<0.01, NS, no significance, versus control groups.