Figure 3.

CK1α promotes PER-DBT interactions to regulate PER stability. A, Drosophila S2 cells were cotransfected with pAc-per-V5-6XHis together with either an empty plasmid (pMT-FH; FH denotes 3XFLAG-6XHis) or pMT-ck1α-FH. Following a 24 h incubation period, kinase expression was induced with CuSO₄ for 16 h. Following induction, CHX was added and cells were harvested for protein extractions at the indicated times (in hours). Proteins were visualized by Western blotting and detected with α-V5. B, Western blots were quantified using ImageLab (Bio-Rad) and normalized against α-HSP70. Error bars indicate SEM from two biological replicates. C, S2 cells were transfected with pAc-per(NLS)-V5-His together with an empty plasmid (pMT-FH), pMT-ck1α(WT)-FH, or pMT-ck1α(K49R)-FH. Equal amounts of the extracted proteins were then incubated with glutathione resin bound with GST or GST-DBT. Bound PER (lanes 4–6) was eluted with 2× SDS loading buffer and visualized via Western blotting in the presence of α-V5. D, Western blots from C were quantified using ImageLab (Bio-Rad) and normalized against input (lanes 7–9). Values were normalized against the PER-NLS sample (without expression of the kinase), which was set as 1. Error bars indicate SEM from four biological replicates. *p < 0.05. E, Head extracts from TUG control or TUG>ck1α RNAi²⁵⁷⁸⁶ flies were prepared and aliquots containing equal amount of total protein for each sample were incubated with glutathione beads bound with GST-DBT. The amount of PER in total head extracts (input) and those that are bound to GST-DBT were visualized by immunoblotting in the presence of α-PER (GP5620). F, Quantification of GST-DBT pull-down assays presented in E. Error bars indicate SEM from four biological replicates. *p < 0.05. G, Head extracts from TUG control or TUG>ck1α RNAi flies were immunoprecipitated with α-PER (GP5620) before Western blotting analysis in the presence of α-PER(pS47) (top) and α-PER (GP5620) (bottom). Asterisk indicates the majority of PER(pS47)-containing isoforms (top). H, Western blots from G were quantified using ImageLab (Bio-Rad) and normalized against the amount of immunoprecipitated total PER. Levels of PER(pS47)-containing isoforms in TUG control were set at 1. Error bars indicate SEM from three biological replicates. **p < 0.005.