Extended Data Fig. 2: Recombinant EPP enzymatic assay and dsRNA silencing efficiencies.
From: A male steroid controls female sexual behaviour in the malaria mosquito
(a) Schematic representation of the reaction expected to be catalyzed by sexually transferred EPP in the mated female atrium. Dotted line highlights the phosphate moiety targeted by EPP. Recombinant EPP (rEPP) catalyzes the dephosphorylation of 3D20E22P in vitro. Using 3D20E22P (extracted from the MAGs by HPLC-MS/MS) as the substrate, HPLC-MS/MS was used to detect 3D20E production following incubation with the recombinant enzyme. DHFR: dihydrofolate reductase, used as negative control; H.I.: heat-inactivated rEPP, used as a second negative control. Data are presented as means ± s.e.m. that were derived from three in vitro assay replicates. (b) Time-course analysis of EPP expression (red) and phosphatase activity (purple) in the MAGs relative to dsGFP at day 0 (means ± s.e.m.). EPP levels were lowest at two days post injection (dpi), and phosphatase activity was lowest at 3–4 dpi. Data were pooled from three replicates. (c) RT-qPCR silencing efficiencies for dsRNA injections, normalized to the housekeeping ribosomal gene RpL19, and calculated as the expression of the target gene relative to the GFP control (means ± s.e.m.). Data were pooled from three replicates.
