Extraction of UDP-GlcNAc and UDP-GalNAc

The procedure for the extraction of UDP-GlcNAc and UDP-GalNAc from Drosophila was optimized using a previously reported method (45). To establish HPLC conditions, we used 10 flies from the w¹¹¹⁸ stain (a standard Drosophila strain). For analysis, we used 25 flies in each sample (1 day old; control and dNGLY1 knockdown, with or without supplementation). Flies were manually ground down in 120 µl of 75% ethanol and cells were further lysed on ice using a sonic dismembrator (Fisher Scientific). After adding additional 300 µl of 75% ethanol, the samples were incubated with a rotator at 4°C for 10 min. Soluble supernatant was collected by centrifugation (14 500 rpm, 20 min at 4°C). The insoluble fractions were extracted a second time if necessary and the combined supernatant was dried using a SpeedVac (LABCONCO). After solvent evaporation, the samples were resuspended in 50 µl of PBS, then diluted to 150 µl using HPLC grade water. A 100-µl of supernatant was filtered through the 3000 MWCO filter (Millipore) and the filtrate was stored at −80°C until ready for further analysis.