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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.
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