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. Author manuscript; available in PMC: 2014 Nov 17.
Published in final edited form as: Curr Biol. 2010 Feb 11;20(4):300–309. doi: 10.1016/j.cub.2009.12.055

Figure 5.

Figure 5

GPRK2 and GR expression levels control rhythms in PrER behavior. (a) Western blot showing GPRK2 expression in proboscises from WT and Gprk206936 mutant (Gprk2 Mut) flies and antennae (Ant) from WT flies. GPRK2 runs as two isoforms in antennae and one isoform in proboscises. ACTIN was used as a loading control (b) Western blot showing GPRK2 levels in proboscises of WT flies collected at the indicated times during an LD cycle. The GPRK2:ACTIN values at ZT1, ZT5, ZT9, ZT13, and ZT21 are relative to the value at ZT17, which was set to 1.0. Each timepoint represents the mean of three independent experiments. The overall effect of time of day is significant (p < 0.005) by one-way ANOVA. (c) GPRK2 and ELAV immunostaining in labellar GRNs from WT and Gprk2 mutant flies. Anti-GPRK2 immunoreactivity is shown in green and anti-ELAV signal is shown in red. Scale bars represent 10 µm. Grey arrows, GPRK2 localization in the cytosol; white arrows, GPRK2 immunostaining in the shaft of a sensillar hair. (d, e) PrER responses to sucrose and trehalose were measured at ZT1 and ZT17 in WT flies carrying Gr5a-Gal4 and UAS-Gprk2, which overexpress GPRK2 in S neurons (GPRK2 OE), and in Gprk206936 mutants (Gprk2 Mut). Mean PrER responses to sucrose (d) and trehalose (e) at ZT1 and ZT17 were not significant (p > 0.16), and remained at constant low levels in Gprk2 Mut flies and constant high levels in GPRK2 OX flies. For each genotype, three or more groups of ≥ 10 flies were tested for PrER responses to sucrose and trehalose at each timepoint. Asterisks denote a significant (p < 0.05) change in PrER responses between ZT17 and ZT1. (f) PrER responses to 100 mM sucrose in Gr64 mutant (R1/+;R2/+;ΔGr64/ΔGr64), Gr64 rescue (R1/+;R2/+;ΔGr64/ΔGr64 carrying one copy of the UAS-Gr64abcd_GFP_f reporter), and Gr64a overexpressing flies at ZT1 and ZT17. The differences in mean responses at ZT1 and ZT17 are not significant in Gr64 mutants (p > 0.90) or Gr64a overexpressing flies (p > 0.05), but significant (p < 0.001) in Gr64 rescue flies. All values are mean +/− S.E.M. As with PrER responses, lower GPRK2 expression in Gprk2 Mut flies disrupts rhythms in GRN spike activity (Fig. S2).