Fig. 1. Protein O-GlcNAcylation oscillates over a 24-h day-night cycle under ad libtum condition.

a Western blots showing daily rhythms in O-GlcNAcylation of nuclear proteins extracted from body tissues of wild type (w¹¹¹⁸) flies fed ad libtum. O-GlcNAcylation is detected using chemoenzymatic labeling in combination with Western blotting using α-streptavidin. Unlabeled samples (middle panel) were processed in parallel to labeled samples (top panel) to identify non-specific signal. Total nuclear proteins were stained using Coomassie blue (bottom panel) and were used for normalization. The asterisk (top panel) denotes non-specific signal. Each biological replication was performed independently with similar results (n = 4). b Quantification of nuclear protein O-GlcNAcylation in (a). Data are double plotted and presented as mean ± SEM (n = 4; p = 0.045; RAIN). α-streptavidin signal of the whole lane was quantified, and the non-specific signal was used for background deduction prior to normalization. Data are double plotted and presented as mean ± SEM. The grey shading indicates the dark phase of each 12 h light:12 h dark (LD) cycle. ZT Zeitgeber time (hr). ZT0 denotes lights-on time.