Image quanta las 4000

Larvae were homogenized with a Dounce tissue grinder and lysed with lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS) containing protease inhibitor cocktail (Roche). Lysates were mixed with 5× Laemmli sampling buffer containing 100 mM DTT and boiled at 95°C for 3 min. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Merck Millipore). After blocking with BLOCKING ONE (Nacalai Tesque), the membranes were incubated with primary antibodies in phosphate-buffered saline containing 0.1% Tween 20 and 10% BLOCKING ONE, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Signals were detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and images were captured by ImageQuanta LAS 4000 (GE Healthcare Bio-Sciences).

All strains were patched on synthetic medium without uracil and allowed to grow overnight. Next day cells from patches were re-suspended and Optical density of culture was measured at 600 nm wavelength using water as blank with the help of spectrophotometer (6133000907, Eppendorf). The following dilutions were prepared 10, 1, 0.1, 0.01 and 0.001 in 96-well plates. In all the growth assays 5 µl of diluted culture was spotted on both SD-URA plates with 2% glucose and 2% galactose. Glucose and galactose plates were imaged at 36–48 h and 60–72 h timeframe respectively using gel documentation system (Image Quanta LAS 4000, GE Healthcare). The settings of camera for imaging were tray position 2, precision setting, 1/30 seconds and brightness at 6.