FIG. 2.

Deletion of dPER aa 755 to 809 strongly attenuates DBT-dependent hyperphosphorylation and degradation of dPER in S2 cells. (A to D) S2 cells were transfected with 600 ng of V5-tagged (A) or nontagged (B to D) versions of dper-containing plasmids (as indicated on top of panels). (A, B, and D) The presence (+) or absence (−) of 200 ng of pMT-dbt-V5 is indicated. Exogenous DBT was induced 36 h after transfection by adding to the media 500 μM CuSO₄ (final). Cells were harvested at the indicated times, and extracts were analyzed by immunoblotting in the presence of either anti-V5 antibodies (A) or anti-dPER (GP73) antibodies (B and D) to visualize DBT or dPER. (B) Cells were either mock treated (−) or incubated (+) with RNAi directed against endogenous slimb (ds-Slimb). (C) Immunoprecipitated dPER(1-1224) (indicated as WT) or dPER(Δ) (indicated as Δ) were incubated with (+) or without (−) 500 units of CKIδ in the presence of [γ-³²P]ATP, and radiolabeled bands were visualized by PAGE and autoradiography, as described in Materials and Methods. •, nonspecific bands. (D) At the top of the panel are shown dPER amino acid sequences from 755 to 809. *, Ser and Thr residues that were mutated to Ala to generate the dPER(S/T9A) version of dPER.