Fig. 2. Amino acid stimulation of the vg gene depends on Na⁺/K⁺-ATPase and V-ATPase activity. Treatment with 10^-6 mol l^-1 bafilomycin A1, a specific inhibitor of vacuolar type H⁺-ATPase, and 10^-5 mol l^-1 ouabain, an inhibitor of Na⁺/K⁺-ATPase, results in a significant reduction of the vg gene expression. (A) Fat bodies of 3- to 5-day-old mosquitoes were dissected and incubated in Aedes physiological saline (APS) with different treatments as indicated. After 1 h the APS was replaced with media containing different combinations of amino acids (AAs), bafilomycin A1 and ouabain, and the fat bodies were incubated at room temperature for 6 h. Total RNA was isolated from three groups of three fat bodies per treatment. cDNA was synthesized from equal amounts of DNase I-treated total RNA. Gene expression was analyzed using vg-specific real-time PCR primers. Values are means ± s.e.m. of triplicate samples. (B) Treatment with bafilomycin A1 or ouabain does not inhibit 20-hydroxyecdysone (20E) activation of the E74 early gene (E74B isoform). As a control, fat bodies were dissected and pre-treated in APS as described above. After 1 h the APS was replaced with media containing different combinations of 10^-6 mol l^-1 20E, 10^-6 mol l^-1 bafilomycin A1 and 10^-5 mol l^-1 ouabain. The fat bodies were incubated at room temperature for 6 h and processed as described above. Gene expression was analyzed using E74-specific real-time PCR primers. Values are means ± s.e.m. of triplicate samples.