Anti dfoxo

Drosophila S2R+ cells were cultured in Corning Insectagro DS2 with 10% FBS (HyClone). Plasmids pUAST-Flag-dFoxO, pUAST-3HA-Pelle and Actin-GAL4 were used for co-transfection as indicated using Effectene Transfection Reagent (QIAGEN). 48 hours after transfection, cells were lysed in RIPA buffer (CST) with PMSF and proceeded with the standard western blot and co-immunoprecipitation protocols. Proteins were probed or immunoprecipitated with following antibodies: Flag-Tag antibody [3A6] (CMCTAG), HA-Tag (C29F4) Rabbit mAb (CST), Anti dFoxO (Cosmo Bio), Anti-rabbit IgG, HRP-linked Antibody (CST), Anti-mouse IgG, HRP-linked Antibody (CST). For Phosphatase Treatment, cell extracts were incubated with calf intestine phosphatase (CIP) (New England BioLabs) for 40 mins at 37°C.

Head, thorax and abdomen in adult fly were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Cat. # 89900) containing the protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Cat. # 78449). The isolated protein samples pooled from 25 flies, were quantified by the Bradford assay and separated in 10% poly-acrylamide gel. The transferred Immobilon^TM PVDF Membrane (Millipore) was blocked for 1 h at room temperature (RT) in TBST containing 5% BSA and incubated overnight at 4°C. The anti-phospho-AKT (Cell signaling, Cat. #4054), anti-pan-AKT (Cell signaling, Cat. #9272), anti-phospho-JNK (Cell signaling, Cat. #9255) anti-dFOXO (Cosmo Bio, Cat. THU-A-DFOXO), and anti-Actin (Cell signaling, Cat. #4967) was diluted to 1:1000 with 5% BSA in TBST when used as a primary antibody. After washing, the membrane was incubated with 5% skim milk in TBST containing HRP-conjugated secondary antibody (1:10000) at RT for 1 h. The HRP signal was generated following the ECL substrate treatment and analyzed by the ChemiDoc mini-HD6 system (UVITEC).