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. Author manuscript; available in PMC: 2012 Apr 29.
Published in final edited form as: Cell. 2011 Apr 29;145(3):357–370. doi: 10.1016/j.cell.2011.04.002

Figure 4. Phospho-specific antibodies reveal that DBT phosphorylates S589 but not S596, and that phosphorylation at S596 stimulates phosphorylation at S589 and S585, which delays phosphorylation at S47.

Figure 4

(A) Extracts were prepared from S2 cells co-transfected with a wild type (WT) version of dper (pAc-dper-V5-His) and pMT-dbt-V5-His (+) or an empty pMT plasmid, pMT-V5-His (−). Cells were treated with MG132 and cycloheximide 16 hours post-dbt induction and harvested 4 hrs later. dPER was immunoprecipitated (IP) with α-V5 beads and split into equal parts that were either treated in the absence (-λPP) or presence (+λPP) of λ-phosphatase. Recovered immune complexes were probed by western blotting (WB) in the presence of the indicated antibody (left of panel). (B) Head extracts were prepared from wper0;p{dper(WT)} or wper0;p{der(Δaa755-809)} flies collected at the indicated times (ZT; top of panels). dPER-HAHis-containing immune complexes were recovered using α-HA beads and split into two equal parts that were treated in the absence (-λPP) or presence (+λPP) of λ-phosphatase. Recovered immune complexes were probed by western blotting (WB) in the presence of the indicated antibody (left of panel). (C, D, E) Head extracts were prepared from wper0;p{dper(WT)}, wper0;p{dper(S589A)}, and wper0;p{dper(S596A)} flies collected at the indicated times (ZT). dPER-HAHis-containing immune complexes were recovered using α-HA beads, followed by western blotting (WB) in the presence of the indicated antibody (left of panel). Positions of non-specific signals from α-pS589 and α-pS47 antibodies are indicated by asterisks (*; right of panels). See Figs. S4 and S5 for supporting data.