Eliminating O-GlcNAcylation at S942 elevates PER-dependent repression of CLK activity in <i>Drosophila</i> S2 cells and in flies.

(A) Repressor activity of PER(WT) and PER O-GlcNAc site mutant variants as measured by per-luc reporter assay in S2 cells (n = 2). The control sample contains CLK in the absence of the repressor PER. Error bars indicate ± SEM. Asterisks denote statistical significance between WT PER and O-GlcNAc site mutants *P P (B) Expression of WT and mutant variants of PER-V5 (Top) and CLK-V5 (Middle) in S2 cells for per-luc reporter assay. HSP70 was detected as loading control (Bottom). (C) Repressor activity of PER(WT) and PER O-GlcNAc site mutant variants as measured by per-luc reporter assay in S2 cells normalized to protein level of PER variants (n = 2). The control sample is not included as PER is not expressed. Error bars indicate ± SEM. Asterisks denote statistical significance between PER(WT) and O-GlcNAc site mutants **P (D,E) Steady state mRNA expression of per and tim in heads of wper0; p{per(WT)-HA10HIS} and wper0;p{per(S942A)-HA10HIS} flies, entrained in 12h:12h LD condition and assayed on LD3 (D) or DD1 (E) (n = 3 biological replicates). Error bars indicate ± SEM (*P-value P-value (F,G) Western blots and corresponding quantifications comparing PER levels between head extracts of wper0; p{per(WT)-HA10HIS} and wper0; p{per(S942A)-HA10HIS} flies on LD3 (F) and DD1 (G). PER-HA levels were detected using both α-PER (GP5620) (Top) and α-HA (Middle). α -HSP70 was used to indicate equal loading and for normalization (Bottom). Three biological replicates were quantified and depicted in graphical format. Error bars indicate ± SEM (*P-value P-value (H,I) Steady state mRNA expression of per (H) and tim (I) in fat body of wper0; p{per(WT)-HA10HIS} and wper0; p{per(S942A)-HA10HIS} flies, entrained in 12h:12h LD condition and assayed on DD1 (n = 3 biological replicates). Error bars indicate ± SEM (*P-value P-value < 0.01).