Drosophila Genetic Manipulation of Insulin Signaling

Drosophila orthologs of human genes were identified via the comparative genomics section of the Ensembl project. TRiP RNAi lines were obtained from Bloomington Drosophila Stock Center. To knockdown genes in adult IPCs, TRiP RNAi lines were crossed to the UAS-Dcr-2.D; Ilp21 gd2HF(attP2) Ilp215–1-GAL4 strain. To knockdown genes in adult fat body, TRiP RNAi lines were crossed to the UAS-Dcr-2.D; Ilp21 gd2HF(attP2) Lk6-GAL4 strain. To measure circulating and total Ilp2HF content per fly, Ilp2HF ELISA^{10 (link)} was used with the following modifications: fifteen ad libitum fed 3-day-old male flies abdomens were dissected open, submerged in 50 μl of PBS with 0.2% Tween 20, and vortexed for 30 min at room temperature. The supernatants of hemolymph extraction were transferred to anti-FLAG antibody coated ELISA plate wells containing 50 μl of anti-HA-peroxidase at 2.5 ng/ml in PBS with Tween 20. Two CPTI protein trap lines for CG9650, CPTI000886 and CPTI001740, were obtained from Kyoto Stock Center. Immunostaining for GFP and Ilp2 protein in adult brains was performed as previously described^{10 (link)} with the following modifications: mouse anti-GFP antibody (1:1000; Invitrogen), rabbit anti-Dilp2 antibody (1:1000) (7), Alexa Fluor 488 and 647 secondary antibodies (1:1000; Invitrogen) were diluted and incubated in PBS with 0.3% Triton-X100.