Ab195977

Protein samples were prepared from larval midguts in lysis buffer containing Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Fisher Cat. No. 78428) and Protease Inhibitor Cocktail (Promega Cat. No. G6521). The following antibodies were used: rabbit anti-pFoxo1 1:1000 (Cat No. 9461, Cell Signaling Technology), rabbit anti-dFoxo1 1:1000 (ab195977, Abcam), rabbit anti-Akt 1:1000 (Cat No. 4691, Cell Signaling Technology), rabbit anti-pAkt 1:1000 (cat No. 4060, Cell Signaling Technology) and mouse anti-actin 1:1000 (DSHB Cat. No. 224236-1). Western blots were developed using Clarify ECL Western Blotting Substrates (BioRad). The bands were detected using an Azure Biosystems c280 digital imager using chemiluminescent detection of HRP. Three independent immunoblots were performed for each experiment.

Late third instar larvae or S2 cells were lysed with lysis buffer (20 mM HEPES pH7.4, 70 mM KCl, 10 mM EDTA, 10 mM EGTA, 2 mM DTT, 1 mM PMSF, 0.1% Igepal CA-630, 16% Glycerol, and Roche EDTA-free Protease inhibitor cocktail) chilled on ice. The lysates were boiled with sample loading buffer at 95 ^°C for 10 mins. The total protein samples were loaded on 7.5% or 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After blocking at RT, membranes were probed sequentially in primary antibody solution and HRP-conjugated secondary antibody solution diluted with 2% dry milk or 2% BSA in TBST (140 mM NaCl; 3 mM KCl; 25 mM Tris pH7.4; 0.1% Tween 20). Pierce ECL western blotting substrate (Thermo) was used to detect immunostained proteins. A cytosolic fraction from salivary glands was isolated as described [35 (link)]. Primary antibodies were rabbit anti-hFOXO1 (1:1000; A2934, Abclonal), rabbit anti-dFoxo (1:1000; ab195977, Abcam), rabbit anti-hTCTP (1:2000; ab37506, Abcam), rabbit anti-Tctp (1:2500) [18 (link)], mouse anti-βTub (1:5000; E7, DSHB), and rabbit anti-GFP (1:10000; ab290, Abcam).