PD98059 inhibited TGF-β1-induced ERK phosphorylation and enhanced TGF-β1-induced <i>NOS</i> expression in the <i>A. stephensi</i> midgut.

<p>(A) PD98059 inhibited ERK activation in mosquito midgut. <i>Anopheles stephensi</i> mosquitoes were fed on artificial bloodmeals supplemented with 2000 pg/ml TGF-β1 with or without 4 or 40 µM PD98059. At 20 min after completion of feeding, 100 midguts from each treatment group were dissected and prepared for immunoblot analysis. Protein lysate concentrations were measured by BCA assay; equal amounts of protein were loaded in each lane. The immunoblot is representative of 3 independent experiments. (B) Inhibition of ERK phosphorylation enhanced TGF-β1-induced <i>NOS</i> expression <i>in vivo</i>. <i>Anopheles stephensi</i> were fed on artificial bloodmeals supplemented with 40 µM PD98059 (PD), 2000 pg/ml TGF-β1, or 40 µM PD98059 and 2000 pg/ml TGF-β1 (TGF-β1+PD). A control group (BM) was provided an identical unsupplemented artificial bloodmeal. Previous studies confirmed that provision of small volumes of PBS and DMSO has no effect on <i>AsNOS</i> expression <i>in vivo</i> (not shown), so these treatments were not included here. <i>AsNOS</i> expression levels were analyzed from midguts (n = 15) collected at 24 h or 48 h after blood feeding from each treatment group. The data are represented as means±standard errors from 5 independent experiments for fold inductions compared with the PBS control. The data were analyzed by ANOVA for overall significance and Student's t-test was used for the pairwise comparison at 24 h.