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. 2008 Apr 7;181(1):65–78. doi: 10.1083/jcb.200712027

Figure 4.

Figure 4.

Bora is ubiquitinated in a β-TrCP1–dependent manner. (A) Identification of the SCF–β-TrCP subunits as Bora-interacting proteins. Listed are peptides of Cul1, Skp1, and β-TrCP1/2 identified by mass spectrometry in the GFP–S-Bora complex together with their XCorr and DeltaCN scores. (B) Bora associates with β-TrCP1 in vivo. HA-β-TrCP1 was cotransfected into HeLa cells with either GFP-Bora (lane 1) or GFP (lane 2) and cells were harvested at 48 h after transfection. Cell lysates and the anti-GFP immunoprecipitates were assayed by Western blotting. (C) Endogenous Bora and SCF–β-TrCP interact during the degradation of Bora. HeLa S3 cells were synchronized as described in Fig. 1 A. Endogenous Bora was immunoprecipitated and associated proteins were analyzed by Western blotting. (D) Bora interacts with β-TrCP1 in a Plk1-dependent manner. TN0 extracts were either mock depleted or depleted of Plk1 as described in Fig. 3 C. In vitro–synthesized 35S-Bora was first incubated with depleted extracts and then with nonlabeled HA-β-TrCP1 that had been immunoprecipitated by the anti-HA antibody/protein A beads. Proteins associated with HA-β-TrCP1 beads were assayed by SDS-PAGE. IP-control corresponds to a control immunoprecipitation of HA-β-TrCP1 with nonspecific IgG. Input, 10% of Bora after incubation with the TN0 extracts. Numbers below lanes 2 and 5 represent β-TrCP1-bound Bora relative to their respective total inputs. (E) β-TrCP1 controls the levels of Bora in vivo. HeLa cells were transfected with HA-β-TrCP1 or HA-β-TrCP1-δF and then synchronized by a thymidine-nocodazole arrest. Mitotic cells were shaken off at 42 h after transfection and protein levels in mitotic cells were determined by Western blotting. (F) β-TrCP1 controls Bora half-life. HeLa cells were synchronized by a double thymidine treatment and transfected with a control siRNA (siControl) or a previously characterized siRNA targeting both β-TrCP1 and 2 (si β-TrCP1/2; Mailand et al., 2006) during the second thymidine arrest. At 8 h after release from the second thymidine arrest, cyclohexamine was added and cells harvested 0, 30, 60, 90, 120, and 150 min later. Half-life of Bora was determined by Western blotting.