Mouse anti mys

Fat bodies from five larvae per sample were dissected in PBS and lysed directly in SDS sample buffer, with three or more biological replicates used for each experiment. Extracts were boiled for 3 min, separated by polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore). Membranes were blocked in PBS + 1% Tween 20 + 5% bovine serum albumin (BSA) and incubated overnight in blocking solution containing primary antibody. Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) with HyBlot CL autoradiography film (Denville Scientific). The antibodies used were rabbit anti-phospho-T398 dS6K #9209 (1:500; Cell Signaling Technology), rabbit anti-phospho-S505 dAkt #4505 (1:1000; Cell Signaling Technology), rabbit anti-phospho-4E-BP1 #2855 (1:1000; Cell Signaling Technology), rabbit anti-phospho-AMPKα #4188 (1:1000; Cell Signaling Technology), mouse anti-β-tubulin E7 (1:1000), mouse anti-mys (1:300; Developmental Studies Hybridoma Bank), rabbit anti-GFP (1:30,000; Molecular Probes, A6455), and rabbit anti-dsRed (1:10,000; Clontech Laboratories, 632496).

Seven to 10 dissected larvae were incubated in M3 + 10 µg/mL insulin medium for 2 h ex vivo, fixed in 4% formaldehyde overnight at 4°C, and washed in PBT. Fat bodies were dissected in PBS and mounted in VectaShield (Vector Laboratories). For Integrin β immunolabeling, carcasses were blocked in PBS + 0.3% Triton X-100 (PBT) + 5% BSA and incubated overnight in blocking solution containing mouse anti-mys (1:100; Developmental Studies Hybridoma Bank). After four washes in PBT, samples were incubated for 2 h in blocking solution containing secondary antibody and washed four times prior to dissection and mounting. Confocal images were collected on a Zeiss LSM700 confocal microscope and processed with Adobe Photoshop. Membrane staining intensity and Pearson correlation coefficient (r) were quantitated using ImageJ.