Plk4 is degraded in a Slimb-dependent manner and is stabilized by perturbing its interaction with Slimb. (A) Plk4 family showing the conserved kinase domain (gray), Polo box motif (striped boxes), and Slimb-binding consensus (black bars). The S293A/T297A SBM should be nondegradable. (B) Preimmune control and anti-Slimb immunoprecipitates from stable S2 cell lysates expressing Plk4-myc were probed for anti-Slimb, SkpA, and myc. (C) Control and anti-GFP immunoprecipitates from S2 cell lysates transiently expressing either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed for endogenous Slimb. IP, immunoprecipitation. (D) Anti-GFP immunoprecipitates from S2 cell lysates transiently expressing triple Flag-ubiquitin and either inducible wild-type Plk4-EGFP or Plk4-SBM–EGFP were probed with anti-GFP (left) and anti-Flag (right) antibody. IB, immunoblot. (E) Anti-GFP immunoblots of lysates prepared from stable SAS-6p–driven Plk4-EGFP that were RNAi treated for the indicated protein for 7 d. (F) Plk4 is phosphorylated. (left) Anti-GFP immunoblots of lysates from control or Slimb-depleted cells transiently expressing inducible Plk4-EGFP. Plk4 accumulates as a doublet (arrowheads) after slimb RNAi. (right) Anti-GFP immunoprecipitates from day 4 Slimb-depleted cells transiently expressing inducible Plk4-EGFP were mock or alkaline phosphatase treated. Plk4 shifts from a broad band (bracket) to a faster migrating single polypeptide (arrowhead). (D–F) Molecular mass is indicated in kilodaltons. (G) Anti-GFP immunoblots showing the levels of transiently expressed SAS-6p–driven Plk4-EGFP and Plk4-SBM–EGFP in 24-h drug-induced cell cycle–arrested cells. Cotransfected Nlp-EGFP was used as a loading control. (H) Anti-GFP immunoblots showing the levels of 4 h–induced Plk4-EGFP and Plk4-SBM–EGFP expression in drug-induced cell cycle–arrested cells. (E and H) α-Tubulin was used as a loading control.