The Drosophila CLOCK Protein Undergoes Daily Rhythms in Abundance, Phosphorylation, and Interactions with the PER–TIM Complex  Choogon Lee, Kiho Bae, Isaac Edery  Neuron  Volume 21, Issue 4, Pages 857-867 (October 1998) DOI: 10.1016/S0896-6273(00)80601-7

Figure 1 Biochemical Detection of dCLOCK in Drosophila Heads (A) Rabbit reticulocyte lysates were programmed with in vitro transcribed RNA as indicated (top). Following in vitro translation in the presence of [35S]methionine, an aliquot of the mixture was resolved by 6% SDS–PAGE and visualized by fluorography and autoradiography. The positions of molecular weight standards (BioRad) are shown at left. (B) Cell-free extracts derived from either in vitro translation reactions (lanes 1–3) or adult fly heads (lanes 4–10) were resolved on 5.7% polyacrylamide gels and transferred to nitrocellulose, and immunoblots were probed with either the anti-dCLOCK antibody GP90 (lanes 1–8) or R13 (lanes 9 and 10). The genotype and time (ZT) of fly collection during a 12 hr light:12 hr dark cycle (LD) are indicated (top of panel). The size range of dCLOCK and a nonspecific band (arrow) are shown at right. Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 2 dCLOCK Undergoes Daily Changes in Abundance that Are Regulated in a Circadian Manner (A) Wild-type flies were collected at the indicated times (ZT) in LD (lanes 1–6). per01 (lane 7) and tim0 (lane 8) flies were collected at several times during the dark phase of LD, and the different time points pooled separately for each genotype. Head extracts were prepared and analyzed by immunoblotting using antibodies directed against dCLOCK (top panels), PER (middle panel), or TIM (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. Arrows (left) indicate nonspecific bands. (B) Quantitation of the relative amounts of dCLOCK (closed square), PER (open triangles), and TIM (open circles) as a function of time in LD. The results for PER and TIM were calculated from the experiment shown in panel (A), whereas those for dCLOCK were derived from three independent experiments. Peak values for each protein were set to 100 and the rest of the values normalized. (C) Comparison of the relative amounts of dClock protein (closed squares) and RNA (open circles) in LD. Peak values were set to 100 and the rest of the values normalized. (D) Wild-type flies were entrained by two cycles of LD, collected at the indicated times (CT) during the first day of constant dark conditions (DD), and treated as described in (A). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 2 dCLOCK Undergoes Daily Changes in Abundance that Are Regulated in a Circadian Manner (A) Wild-type flies were collected at the indicated times (ZT) in LD (lanes 1–6). per01 (lane 7) and tim0 (lane 8) flies were collected at several times during the dark phase of LD, and the different time points pooled separately for each genotype. Head extracts were prepared and analyzed by immunoblotting using antibodies directed against dCLOCK (top panels), PER (middle panel), or TIM (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. Arrows (left) indicate nonspecific bands. (B) Quantitation of the relative amounts of dCLOCK (closed square), PER (open triangles), and TIM (open circles) as a function of time in LD. The results for PER and TIM were calculated from the experiment shown in panel (A), whereas those for dCLOCK were derived from three independent experiments. Peak values for each protein were set to 100 and the rest of the values normalized. (C) Comparison of the relative amounts of dClock protein (closed squares) and RNA (open circles) in LD. Peak values were set to 100 and the rest of the values normalized. (D) Wild-type flies were entrained by two cycles of LD, collected at the indicated times (CT) during the first day of constant dark conditions (DD), and treated as described in (A). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 2 dCLOCK Undergoes Daily Changes in Abundance that Are Regulated in a Circadian Manner (A) Wild-type flies were collected at the indicated times (ZT) in LD (lanes 1–6). per01 (lane 7) and tim0 (lane 8) flies were collected at several times during the dark phase of LD, and the different time points pooled separately for each genotype. Head extracts were prepared and analyzed by immunoblotting using antibodies directed against dCLOCK (top panels), PER (middle panel), or TIM (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. Arrows (left) indicate nonspecific bands. (B) Quantitation of the relative amounts of dCLOCK (closed square), PER (open triangles), and TIM (open circles) as a function of time in LD. The results for PER and TIM were calculated from the experiment shown in panel (A), whereas those for dCLOCK were derived from three independent experiments. Peak values for each protein were set to 100 and the rest of the values normalized. (C) Comparison of the relative amounts of dClock protein (closed squares) and RNA (open circles) in LD. Peak values were set to 100 and the rest of the values normalized. (D) Wild-type flies were entrained by two cycles of LD, collected at the indicated times (CT) during the first day of constant dark conditions (DD), and treated as described in (A). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 2 dCLOCK Undergoes Daily Changes in Abundance that Are Regulated in a Circadian Manner (A) Wild-type flies were collected at the indicated times (ZT) in LD (lanes 1–6). per01 (lane 7) and tim0 (lane 8) flies were collected at several times during the dark phase of LD, and the different time points pooled separately for each genotype. Head extracts were prepared and analyzed by immunoblotting using antibodies directed against dCLOCK (top panels), PER (middle panel), or TIM (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. Arrows (left) indicate nonspecific bands. (B) Quantitation of the relative amounts of dCLOCK (closed square), PER (open triangles), and TIM (open circles) as a function of time in LD. The results for PER and TIM were calculated from the experiment shown in panel (A), whereas those for dCLOCK were derived from three independent experiments. Peak values for each protein were set to 100 and the rest of the values normalized. (C) Comparison of the relative amounts of dClock protein (closed squares) and RNA (open circles) in LD. Peak values were set to 100 and the rest of the values normalized. (D) Wild-type flies were entrained by two cycles of LD, collected at the indicated times (CT) during the first day of constant dark conditions (DD), and treated as described in (A). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 3 dCLOCK Is Phosphorylated throughout a Daily Cycle Head extracts prepared from wild-type flies collected at the indicated times during LD (ZT) were incubated with anti-dCLOCK antibodies and immune complexes recovered by centrifugation. Immune complexes ([A] lanes 1–6, [B] lanes 1 and 2) and reticulocyte lysates containing in vitro translated dCLOCK ([A] lanes 7–9, [B] lane 3) were incubated in the presence (+) or absence (−) of lambda protein phosphatase (λPPase) and vanadate as indicated. Following incubation, the mixtures were subjected to immunoblotting in the presence of anti-dCLOCK antibodies. The positions of the different mobility variants of dCLOCK are indicated (left). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 4 PER and TIM Interact with dCLOCK in a Time-of-Day-Specific Manner Wild-type flies were collected at the indicated times (ZT) in LD. per01 (lane 8) and tim0 (lane 9) flies were collected at several times during the dark phase of LD and the different time points pooled separately for each genotype. Head extracts were prepared and subjected to immunoprecipitation using antibodies against dCLOCK. Immune complexes were recovered by centrifugation and analyzed by immunoblotting using antibodies directed against dCLOCK (top panel), PER (middle panel), or TIM (bottom panel). Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)

Figure 5 PER, TIM, and dCLOCK Are Present in the Same Complex (A) Wild-type flies were collected at the indicated times (ZT) in LD and head extracts incubated with antibodies against PER or TIM as indicated (+). Immune complexes were recovered by centrifugation and analyzed by immunoblotting using antibodies directed against PER (top panel), TIM (middle panel), or dCLOCK (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. (B) Head extracts were prepared from wild-type flies collected at ZT20 and subjected to sequential immunoprecipitation (IP) reactions. Anti-PER antibodies were first added and immune complexes recovered by centrifugation (lane 1). The supernatant was subsequently transferred to a fresh tube and subjected to a second IP reaction using anti-TIM antibodies (lane 2). The immune complexes from both IP reactions were analyzed by immunoblotting using antibodies directed against PER (top panel), TIM (middle panel), or dCLOCK (bottom panel). The size ranges of dCLOCK, PER, and TIM are shown at left. (C) Head extracts were prepared from the 13.2per–HA10His transgenic flies collected at ZT23.9. PER–HA10His was purified using metal affinity chromatography and subjected to immunoprecipitation in the presence of anti-TIM antibodies. The immune complex was analyzed by immunoblotting using antibodies directed against dCLOCK (lane 1), PER (lane 2), or TIM (lane 3). The arrow identifies a nonspecific band. Neuron 1998 21, 857-867DOI: (10.1016/S0896-6273(00)80601-7)