Western blots of early phase (AeET) and late phase (AaSPVI, AaSPVII, AaLT) protease expression in unfed and blood fed dsRNA injected mosquitoes were performed as previously described [31] . Analysis of KDEL containing proteins by Western blotting was done by dissecting fifty midguts from amino acid fed or unfed (sugar fed) female mosquitoes in pre-cold PBS buffer, dipped into 76Protease Inhibitor Cocktail in 100 mM phosphate buffer (pH 7.0) (Roche Applied Science, Germany) with a forceps, and transferred into an ice-cold 1.5-ml Eppendorf tube containing 60 ml of PBS/TDS buffer (1% Triton X-100, 12 mM Na deoxycholate, 0.2% SDS in PBS, and Protease Inhibitor Cocktail). The dissected midguts were homogenized using a blue Kontes pestle. The homogenate was incubated on ice for 10 min and then spun at 10000 rpm for 10 min at 4uC. The supernatant was transferred to a pre-chilled 1.5-ml tube and 56SDS sample loading buffer was added. The mixture was boiled for 4 min, chilled on ice for 2 min, and then spun at 13000 rpm for 5 min at room temperature. The yielded supernatant was stored at 220uC for SDS-PAGE.