Figure 5.

Altered circadian protein expression in kay-αRNAi flies. A, KAY-α downregulation alters expression of several circadian proteins. Flies were entrained to a standard LD cycle for 3 d and then released in DD. Fly brains were dissected on the first day of DD at the expected peak time point of the protein measured, followed by immunocytochemistry (kay-αRNAi flies were dissected ∼1–1.5 h after control flies to correct for differences in period length). Representative staining images and quantifications are shown. Arrows point to sLNvs in the focal plane. Quantification of protein levels are represented by boxes and whiskers in which whiskers show the minimum and maximum values, boxes show the middle 50% of the values, and horizontal lines in the boxes show the median. Two to four independent experiments were performed. CLK, PER, and PDP1 levels were markedly reduced in kay-αRNAi in all experiments. VRI levels were unaffected. TIM levels were reduced in kay-αRNAi flies in all three experiments, but only in one experiment was that decrease statistically significant. Student's t test. ***p < 0.001; n.s., not significant. B, KAY-α regulation of PER levels is strongly dependent on the per promoter. When PER was expressed independently of its promoter with Pdf–GAL4 in a per⁰ background, KAY-α downregulation had weak effects on PER levels. Quantifications of two independent experiments are shown. In the first one, the average 13% decrease in PER level in kay-αRNAi flies was not statistically significant, but the average 22% decrease in the second experiment was marginally significant (Student's t test, p = 0.04). Thus, posttranscriptional regulation of PER might have a weak contribution to its protein decrease in kay-αRNAi flies.