Fig. 2. Clvs2 participates in 4VA signal transduction downstream of OR35.
(A) Structure model of Clvs2 protein. α helices are shown in orange, loops in gray, and β barrel in light blue. (B) Binding affinity of Clvs2 protein to 4VA was determined using the MST analysis (n = 3). The solid curve was fit to the standard KD-fit function. (C) Clvs2 expression following 4VA exposure and Or35 RNAi (n = 7). “+” and “−” symbols below each bar indicate the presence and absence of the corresponding treatment, respectively: dsRNA injection (dsGFP or dsOr35) and odorant exposure (oil or 4VA). (D) OR35 (magenta) and Clvs2 (green) colocate under basiconic sensilla in antennae of gregarious locusts. NC, negative control; BF, bright field of image. (E) Immunofluorescence labeling of recombinant fusion OR35-mOrange and Clvs2-eGFP in HEK293T cells. Cells transfected with the empty pcDNA3.1(+) plasmid were used as negative controls. Scale bars, 10 μm. (F) Immunofluorescence localization of recombinant fusion Clvs2-eGFP in HEK293T cells. Cytolemma were counterstained with DiI, and nuclei were counterstained with Hoechst 33342. Scale bars, 10 μm. (G) Electroantennography (EAG) responses of antennae to 4VA in gregarious locusts with Clvs2 RNAi knockdown. (H) Representative spike traces of basiconic sensilla to 4VA after RNAi of Clvs2. (I) Responses of basiconic sensilla to 4VA at different concentrations in gregarious locusts with Clvs2 RNAi knockdown. Gene expressions were analyzed by the one-way analysis of variance (ANOVA). The comparisons of electrophysiological recordings were analyzed by the two-tailed unpaired t test [(G) and (I)]. Data in [(G) and (I)] are presented as the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.