Adult worms were allowed to lay eggs for 12 hr on plates seeded with E. coli HT115 transformed with RNAi plasmids. The resulting progeny were themselves allowed to develop to young adults at 20°C before harvesting in M9 buffer. Worms were then placed either on control plates or plates containing 1 mM sodium azide for 2 hr at 20°C [25] (link). Sodium azide plates were made by pipetting a stock solution directly onto NGM plates seeded with E. coli OP50 72 hr before use.
Following treatment worms were washed off plates using M9, collected by settling and resuspended in 100 µl Phosphosafe extraction reagent (Millipore) supplemented with a protease inhibitor cocktail (Roche). Protein concentration was determined using BCA (Pierce) and 40 µg protein was separated by SDS-gel electrophoresis. Proteins were subsequently transferred to nitrocellulose membrane and probed using a 1∶1000 dilution of phospho-AMPKα (Thr172) antibody (Cell Signaling). β-actin antibody (Santa Cruz) was used as a loading control. Imaging and quantification of bands was carried out using the ImageQuant LAS4000 imaging system and software (GE Healthcare).
Following treatment worms were washed off plates using M9, collected by settling and resuspended in 100 µl Phosphosafe extraction reagent (Millipore) supplemented with a protease inhibitor cocktail (Roche). Protein concentration was determined using BCA (Pierce) and 40 µg protein was separated by SDS-gel electrophoresis. Proteins were subsequently transferred to nitrocellulose membrane and probed using a 1∶1000 dilution of phospho-AMPKα (Thr172) antibody (Cell Signaling). β-actin antibody (Santa Cruz) was used as a loading control. Imaging and quantification of bands was carried out using the ImageQuant LAS4000 imaging system and software (GE Healthcare).