FIG 5.

gp83 inhibits Toll signalling downstream of Dif and dorsal. (A) The ability of gp83 to inhibit endogenous Drosomycin expression was assessed by transfection of S2 cells with pAc-gp83 or empty control plasmid, and the Toll pathway was activated by cotransfection of pAc-Toll^LRR or control plasmid. Drosomycin expression levels were measured relative to Rpl32 expression by qRT-PCR. (B to E) The Toll pathway was activated downstream of the Toll receptor by transfection of a plasmid encoding pll (B), knockdown of cactus with two independent, nonoverlapping dsRNAs (labeled ds-cact¹ and ds-cact²) (C), and transfection of plasmids encoding the transcription factors dl and Dif (D and E). Activation of the pathway was assessed using the Drs-FLuc reporter, relative to RLuc expression (orange bars in panels B to E; gray bars represent controls in which empty plasmids [B, D, and E] or dsRNA targeting GFP [C] were transfected). Suppression of the Toll pathway at different stages by gp83 was assessed by cotransfection of pAc-gp83 or an empty control plasmid (B to E). (F) Representative confocal image of S2 cells expressing V5 epitope-tagged gp83 stained with a V5 antibody (top) and a merged image in which nuclei are stained with Hoechst (bottom). Error bars show SEMs (n = 5 biological replicates).