Clarify ecl western blotting substrates

Proteins were extracted from larval midguts in lysis buffer containing phosphatase cocktail (Thermo Fisher Cat. No. 78428) and protease inhibitor cocktail (Promega Cat. No. G6521). The following antibodies were used: rabbit anti-pAMPKα 1:1000 (Thr172) (CST Cat. No. 2531), rabbit anti-AMPKα 1:1000 (CST Cat. No. 2532), mouse anti-actin 1:1000 (DSHB Cat. No. 224-236-1), mouse anti-tubulin 1:1000 (Santa Cruz Biotechnology Cat# sc-8035), goat anti-rabbit-HRP and goat anti-mouse-HRP 1:2000 (Jackson ImmunoResearch Laboratories). 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. At least three independent immunoblots were performed for each experiment.

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.