Locomotor activity of <i>Cul-3</i> downregulated flies in LD and DD.

<p>Control flies and flies expressing either a <i>Cul-3</i> RNAi (A) or a CUL-3^K717R protein (B) were entrained for 4 d in LD 12∶12 and transferred to DD. White and black/gray indicate lights-ON and lights-OFF, respectively. ZT is Zeitgeber Time (ZT0 corresponds to lights-ON). Top panels: averaged activity distribution of <i>n</i> flies in LD (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001367#s4" target="_blank">Materials and Methods</a>). Dots indicate the s.e.m. of the activity for each 0.5-h interval. Average activity per 0.5-h is indicated in parentheses on the left. Bottom panels: averaged actograms during both LD and DD conditions (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001367#s4" target="_blank">Materials and Methods</a>). Behavioral analyses were repeated three to four times with very similar results.</p

TIM interactions with CUL-3 and SLMB.

<p>(A) Anti-CUL-3-FLAG immunoprecipitation from head extracts followed by anti-TIM immunoblotting in <i>per⁺</i> (left) and <i>per⁰</i> (right) backgrounds. Slots 1 and 3 are negative controls (faint band reveals low non-specific anti-FLAG background), and 50 µg of total proteins were used for <i>per⁺</i> and <i>per⁰</i> input extracts. Flies were entrained 3 d in LD and transferred to DD for collection at CT15. (B) Left: anti-FLAG-SLMB immunoprecipitation from head extracts collected at different time points, followed by anti-TIM immunoblotting. Two consecutive time points were pooled for IPs in a <i>per⁺</i> background. Slot 7 is the negative control, and 50 µg of extracts were used for <i>per⁺</i> input. Right: anti-TIM Western blot of head extracts of <i>w; tim-gal4</i> (50 µg) and <i>per⁰ w; tim-gal4</i> (25 µg) indicate that TIM levels are much higher in a <i>per⁰</i> background. At least two independent experiments were done for each assay with very similar results.</p

PER and TIM in head extracts of flies expressing CUL-3^K717R or <i>Cul-3</i> RNAi.

<p>Flies were entrained for 3 d in LD, then transferred to DD. Gray and black bars indicate subjective day and subjective night, respectively. (A) TIM (top, Tris-acetate gel) and PER (bottom, Tris-glycine gel) Western blot analysis of head extracts from flies collected the first and second days of DD. Phosphorylated (P-) and hypo-phosphorylated forms of PER and TIM are indicated. (B) TIM (left) and PER (right) Western blots (both Tris-glycine gels) of head extracts from flies collected the second day of DD. Two (<i>Cul-3RNAi</i>) or three (<i>Cul-3^K717R</i>) independent Western blots were done for each condition with very similar results.</p

PER and TIM proteins in flies <i>Cul-3^K717R</i> and <i>slmb^m</i> flies.

<p>(A) Anti-TIM (top) and anti-PER (bottom) Western blots of head extracts. Flies were entrained 3 d in LD and transferred to DD for collection during the first and second day of DD. Gray and black bars indicate subjective day and subjective night, respectively. (B) Anti-PER Western blots of head extracts. Flies were entrained 3 d in LD and collected the fourth day. White and black bars indicate day and night, respectively. (C, D) Anti-PER (C) and anti-TIM (D) Western blots of head extracts <i>tim⁰</i> and <i>per⁰</i> backgrounds, respectively. Flies were entrained for 3 d in LD and transferred to DD for collection at CT27. Vertical dashes indicate that the two surrounding slots were not contiguous on the gel. Two to three independent Western blots were done for each condition with very similar results.</p

PER and TIM in the s-LNvs of flies expressing <i>Cul-3</i> RNAi.

<p>Flies were entrained for 3 d in LD and collected every 4 h on the fourth day of LD (A) or transferred to DD and collected on the second (B) or third (C) day of DD. Images are optical sections of individual PDF-expressing s-LNvs from <i>Pdf>RNAi</i> flies and controls in LD. Merged images: red is PER, blue is TIM, and green is PDF. Graphs represent quantifications of PER and TIM immunolabeling (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001367#s4" target="_blank">Materials and Methods</a>) in the PDF-positive s-LNvs of <i>Pdf>RNAi</i> or <i>tim>Cul-3^K717R</i> flies and relevant controls. White and gray bars indicate lights-ON and lights-OFF, respectively. Gray and black bars indicate subjective day and subjective night, respectively. Time (h) is indicated as ZT or CT (Circadian Time) where CT0 is 12 h after lights-OFF of the last LD day. Error bars indicate s.e.m. Two independent experiments were done for each genotype/condition with very similar results.</p

Locomotor activity rhythms in constant darkness of flies with altered CUL-3 activity.

<p>The mean values of period and associated power (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001367#s4" target="_blank">Materials and Methods</a>) are given ± s.e.m. In genotypes with altered CUL-3 activity, rhythmic flies display weak rhythms as indicated by the large s.e.m. of the period and the low associated power.</p

PER and TIM proteins in <i>per</i> or <i>tim</i> mutants expressing CUL-3^K717R.

<p>Flies were entrained for 3 d in LD, transferred to DD, and collected at CT27. (A) anti-PER (top) and anti-TIM (bottom) Western blots of head extracts in a <i>tim⁰</i> and <i>per⁰</i> backgrounds, respectively. (B) Optical section of an individual s-LNv immunolabeled for TIM (blue) and PDF (red) in a <i>per⁰</i> background. (C) Anti-TIM Western blot of head extract in a <i>tim⁰ tim^Δ260–292</i> background. Vertical dashes indicate that the two surrounding slots were not contiguous on the gel (A) or that a different exposure was used (C) (shorter exposure for <i>per⁰</i> extracts, which have more TIM). At least two independent experiments were done for each assay with very similar results.</p

<i>per</i> and <i>tim</i> mRNA oscillations in flies expressing CUL-3^K717R.

<p>Flies were entrained for 3 d in LD and transferred to DD for collection every 3 h in the second (left) and third (right) days of DD. Relative <i>per</i> and <i>tim</i> mRNA levels in head extracts were determined by quantitative RT-PCR (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001367#s4" target="_blank">Materials and Methods</a>). For each experiment, the averaged normalized values of both mutant and control genotypes were expressed as a percentage of the maximum value set to 100. Means of two independent experiments are reported in the graphs. Error bars indicate the range between the two experimental values.</p

CK2β does not contribute to the inhibition of CLK degradation.

<p>(A, B) Western blot of nonsonicated head extracts from flies collected at DD1. Gray and black bars represent subjective day and subjective night, respectively. A CB stained band in the size range of CLK is used as a loading control. Brackets indicate hypo- and hyperphosphorylated forms of CLK. At least two independent experiments were performed for each blot. (A) Comparison between <i>tim > CkIIβ RNAi</i> (<i>w; tim-gal4/106845; 32377/+</i>) and <i>tim-gal4/+</i> controls in a <i>per⁺</i> background, for TIM, PER, and CLK proteins. (B) Comparison between <i>tim > CkIIβ RNAi</i> and <i>tim-gal4/+</i> controls in a <i>per⁰</i> background, for TIM and CLK. <i>a</i> and <i>b</i> are different protein extracts from the same genotype at the same time point. (C) Quantitative RT-PCR measurements of <i>Clk</i> mRNA levels in heads of flies collected at DD1. Results shown are means of pooled values from two time points (CT2 and 14, which gave similar values) with two independent samples for each time point. Error bars indicate s.e.m. Average values were normalized to the mean control (<i>per⁰ w</i>; <i>tim-gal4</i>/+) values set to 100. (D) CLK protein/<i>Clk</i> mRNA ratio calculated from mean CT2–CT14 values of Western blot (B) and quantitative RT-PCR (C) data. Ratios were normalized to the control (<i>per⁰ w</i>; <i>tim-gal4</i>/+) ratios set to 100.</p

PER, TIM, and CLK are found in protein complexes containing CK2.

<p>Anti-FLAG immunoprecipitation (IP) from nonsonicated head extracts of flies collected at DD1. FLAG-CK2α or FLAG-CK2β was immunoprecipitated from 1 mg of total protein extracts from heads and 50% of the precipitate was subjected to Western blot analysis. We loaded 50 µg of head extracts as input controls. IgG LC indicates the immunoglobulin G light chain used for the precipitation that is well detected by the anti-mouse secondary antibody. Gray and black bars represent subjective day and subjective night, respectively. Experiments were performed at least twice. (A, Left) FLAG-CK2α was immunoprecipitated from <i>tim >FLAG-CkIIα</i> flies and <i>tim-gal4</i>/+ negative controls (control) in a <i>per⁺</i> background. IP of CK2α (FLAG) and co-IP of CK2β, TIM, PER, and CLK were visualized by immunoblotting. «*» shows an aspecific band recognized by the anti-PER antibody. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs on <i>per⁺</i> background. The time “CT10” indicates a mixed population of flies harvested at CT8 and CT12. A CB stained band in the size range of CLK is used as a loading control. TIM was run on a 3–8% Tris-Acetate gel. (B, Left) FLAG-CK2β was immunoprecipitated from <i>tim > FLAG-CkIIß</i> flies and <i>tim-gal4</i>/+ negative controls (control) in a <i>per⁺</i> background. IP of CK2ß (with anti-CK2ß) and co-IP of CK2α, TIM, PER, and CLK were visualized by immunoblotting. (Right) TIM, PER, and CLK immunoblots of the corresponding inputs. A CB stained band in the size range of CLK is used as a loading control. (C) FLAG-CK2α or FLAG-CK2β was immunoprecipitated from <i>tim >FLAG-CkIIα</i> (<i>α</i>) flies and <i>tim-gal4</i>/+ negative controls (C) in a <i>per⁰</i> background. co-IP of CLK was visualized by immunoblotting. Input samples and immunoprecipitates were run on the same gel for <i>C</i> and <i>α.</i> (D) Image showing PDF (green), CK2α (magenta), and CLK (blue) fluorescent immunolabeling in small PDF⁺ LNv-s of an adult fly at ZT3. The fourth square is a composite picture of the three stainings. Single optical planes are shown taken by confocal microscopy.</p