Rabbit anti phospho t398 ds6k

Fat bodies from 5 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 3 minutes, separated by polyacrylamide gel electrophoresis, and transferred to Immobilon-P membranes (Millipore, Billerica MA). Circulating Dilp3 levels were determined from hemolymph of 10 larvae per sample, diluted 1:100 in PBS and spotted (1 uL) onto methanol-soaked Immobilon-P membranes (Millipore, Billerica MA). Air-dried membranes were blocked in PBT + 5% BSA, and incubated overnight in blocking solution containing primary antibody. Signals were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL) with BioMax Light (Kodak, Rochester NY) or HyBlot CL autoradiography film (Denville Scientific, Metuchen NJ), and quantitated using Adobe Photoshop software. Antibodies used were rabbit anti-phospho-T398 dS6K (1:250), rabbit anti-phospho-S505 dAkt (1:1,000), (both from Cell Signaling Technology, Beverly MA), rabbit anti-Dilp3 (1:1,000; gift of J. Veenstra, Université Bordeaux, Talence, France, ref. 34 (link)), and mouse anti-beta-tubulin E7 (1:1,000; Developmental Studies Hybridoma Bank, Iowa City IA).