About 40 midguts from the fed or unfed female mosquitoes were dissected with 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 Figure 9 . A deficiency in alpha-COPI inhibits feeding-induced expression of late phase midgut proteases. A) Representative Western blot of AeET protein expression in the midguts of mosquitoes injected with 400 ng of Fluc or alpha-COPI dsRNA and maintained on sugar for 3 days and then dissected (unfed), or blood fed after 3 days of sugar feeding and dissected at 3 hr PBM (fed). The GAPDH antibody was used as a protein loading control. Each lane contains the same midgut equivalents obtained from pooled mosquitoes. B) Representative Western blots of protein extracts prepared from pooled mosquito midguts dissected at 24 hr PBM and analyzed with antibodies against late phase serine proteases (AaSPVI, AaSPVII, AaLT) or GAPDH. Mosquitoes were injected with the indicated amount of dsRNA 3 days prior to blood feeding. doi:10.1371/journal.pone.0018150.g009