PER O-GlcNAcylation levels show correlation with feeding activity.

(A) Western blot showing PER O-GlcNAcylation profile over a circadian cycle. PER proteins were immunoprecipitated by α-HA from head extracts of wper0; p{per(WT)-HA10HIS} flies entrained in 12h:12h LD cycle. O-GlcNAcylated PER was chemoenzymatically labeled with 20-kDa PEG mass tag, resolved by SDS-PAGE, and immunoblotted using α-PER (GP5620) (Top). Slower migrating bands, denoted by asterisks, are O-GlcNAcylated PER while non-glycosylated PER migrates faster. Input for immunoprecipitation and labeling were also directly probed for PER (Bottom). (B) PER proteins were immunoprecipitated by α-HA from head extracts of wper0; p{per(WT)-HA10HIS} flies entrained in 12h:12h LD cycle. O-GlcNAcylated PER was chemoenzymatically labeled with biotin, resolved by SDS-PAGE, and immunoblotted using α-streptavidin (Top) and α-PER (GP5620) (Middle). Input for α-HA IP was immunoblotted with α-PER (Bottom). (C) Quantification of PER O-GlcNAcylation over a circadian cycle as shown in Fig 6B (n = 2). Normalization was performed using the signal for α-HA IP for PER-HA. Error bars = SEM. Rhythmicity of PER O-GlcNAcylation was confirmed by JTK-cycle (P < 0.0005).