Fig. 2

GCN1 is expressed in reproduction-associated tissues, and its signaling activity is reduced after RNAi-mediated knockdown. a qRT-PCR profiling of GCN1 expression in adult tissues (top) and western blotting of GCN1 protein expression (bottom) revealed expression of GCN1 in ovaries, fat body and Malpighian tubules. A one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test was used to determine significant differences (P < 0.05) in gcn1 transcript expression. A 15-µg aliquot of protein from each sample was loaded for western blot detection. b Western blot of GCN1 expression after dsRNA injection. c Percentage GCN1 expression relative to GFP dsRNA treatment shows similar decrease in GCN1 expression at 3- and 5-days post-injection (PI), with an increase in relative GCN1 expression beginning by 7 days PI. Pixel intensity from GCN1 bands in 2b (293 kDa, arrow) was standardized using the pixel intensity of the bright approximately 50-kDa band (arrowhead), which had similar intensities across all samples. Total protein was extracted from pools of 10 whole mosquitoes for each sample (n = 1) and 15 µg of protein from each sample was analyzed. d Representative western blots of phospho-eIF2α (p-eIF2α), eIF2α, and actin from three pools of five female abdomens (n = 3), with midguts removed, after injection with either GCN1 dsRNA or green fluorescent protein (GFP) dsRNA. Injected mosquitoes were sampled at different time points post-blood meal (PBM): 0 (unfed), 6, 12 or 24 h PBM. A 10-µg aliquot of total protein from each sample was loaded. e Pixel intensities of p-eIF2α bands from GCN1-injected and GFP-injected mosquitoes in blot from 2d was normalized to pixel intensity of eIF2α bands from 2d to plot change in p-eIF2α band intensity at different time points PBM. Letters represent statistical significance groups (P < 0.05) as determined by analysis with a one-way ANOVA followed by Tukey’s HSD post hoc test for significant differences between treatment groups and time points. FB, fat body; HE, head; OV, ovary; MG, midgut; TH, thorax; MT, Malpighian tubule; WM, whole mosquito; for other terms, see Abbreviations