Figure 2.
PER stably associates with DBTAR but is mostly hypophosphorylated. A, Western blots of DBT protein from adult head extracts of control wild-type flies (WT; ZT23; left) and tim01; dbtar double-mutant flies (right). DBT+ and DBTAR proteins accumulated to similar levels. The result shown here is representative of three experiments and is consistent with the results of Rothenfluh et al. (2000c). B, An affinity-purified anti-DBT antibody was used to immunoprecipitate (IP) DBT from head extracts of wild-type flies (ZT23; left lane) and tim01; dbtar double-mutant flies (right lane). Immunoprecipitates were then subjected to Western blotting with anti-PER (top) and anti-DBT (bottom) antibodies. PER was immunoprecipitated by anti-DBT antibodies in both wild-type and tim01; dbtar double-mutant flies. The results shown are representative of three experiments. C, Head extracts of wild-type flies from ZT14 (left lane) and ZT2 (middle lane) were run alongside extracts from tim01;dbtar double-mutant flies (right lane) and blotted for PER protein. Because tim01; dbtar double-mutant flies are arrhythmic, they were not entrained. PER shows a characteristic difference in mobility in wild-type flies between ZT14 and ZT2, with the decreased mobility at ZT2 attributable to increased PER phosphorylation (Edery et al., 1994). Neither of these times correspond to peak PER levels in wild-type flies but were chosen as extremes in PER phosphorylation status. PER protein extracted from tim01; dbtar double-mutant flies was mostly hypophosphorylated, although some slower mobility PER was detected. The results shown are representative of three experiments.