Fig. 7

RNAi ecdysone receptor (EcR) knockdown effects on AaCHYMO protease expression and activity. (A) Western blot analysis of the EcR knockdown effect on AaCHYMO protein expression using an anti-AaCHYMO primary antibody. Midgut protein extracts were prepared from individual mosquitoes microinjected with dsRNA (RNAi-FLUC and -AaCHYMO). Each lane represents the set number of mosquito samples used for analysis. Each lane contains 0.5 midgut tissue equivalent. The Western blot is representative of 3 independent biological replicates. α-tubulin was used as an internal control. The WB was cropped for clarity. (B) The AaCHYMO bands detected by western blot (A) were quantified and normalized to α-tubulin using NIH Image J software. Data represents the mean ± SEM. **** p < 0.0001 determined using the unpaired Student’s t-test when compared to dsRNA-FLUC controls. (C) Quantification of AaCHYMO mRNA levels in EcR knockdown mosquito tissue extracts compared to the FLUC control. The experiment was conducted from 3 biological cohorts using 12 mosquitoes. Data are presented as mean ± SEM. Statistical significance is represented by stars above each comparison (unpaired Student’s t-test; **** p < 0.0001 compared to the RNAi-FLUC control samples). (D) Chymotrypsin-like activity comparison between RNAi EcR and the FLUC control midgut extract samples using the MeO-Suc-Arg-Pro-Tyr-AMC substrate. Significant reduction (****p < 0.0001) in overall activity is observed in the EcR knockdown samples (3 to 4 biological cohorts using 3 mosquitoes were tested). Please note that the original blot in (A) is presented in Supplementary File 1.