Endo h and pngase f

For Western blotting, fly heads were homogenized in SDS sample buffer with a pellet pestle (Fisher, Pittsburgh, PA). Proteins were then fractionated by SDS–PAGE and transferred to Immobilon-FL transfer membranes (Millipore, Carrigtohill, Ireland) in Tris-glycine buffer. The blots were probed with primary antibodies against Rh1 (mouse, 1:2000; Developmental Studies Hybridoma Bank, Iowa City, IA) or INAD (rabbit, 1:2000, Wang et al., 2008 ), followed by IRDye 680 goat anti-mouse immunoglobulin G (IgG) or IRDye 800 goat anti-rat IgG (Li-Cor, Lincoln, NE) as secondary antibodies. Signals were detected using an Odyssey infrared imaging system (Li-Cor, Lincoln, NE).
All reagents for the Endo H and PNGase F digestions were obtained from New England Biolabs (NEB, Ipswich, MA). Twenty fly heads were collected and homogenized in glycoprotein denaturing buffer (1% SDS, 80 mM dithiothreitol). Homogenates were incubated for 4 h at 22°C and centrifuged to collect the supernatant. Digest reactions were processed according to manufacturer’s instructions with slight modification. Specifically, the reactions were incubated for 4 h at 37°C. All samples were solubilized in SDS loading buffer to equal volumes for Western blot analysis.