Figure 2.
Figure 2. Asl contains a second Plk4 PB1-PB2–binding domain in its C terminus that is sufficient for stabilizing Plk4. (A) Asl stabilizes Plk4 primarily through its C terminus. The relative stability of Plk4-EGFP protein was analyzed by immunoblotting lysates of S2 cells transiently coexpressing the indicated inducible Asl-V5 constructs. Cotransfected Nlp-EGFP was expressed under its endogenous promoter and served as a loading control. Blots were probed with anti-GFP, V5, and Asl. In lane 2, FL Asl-V5 and endogenous (Endo) Asl are not resolvable, so the band contains both species. (B) Asl stabilizes Plk4 in cells lacking centrioles. S2 cells were control or SAS-6 RNAi treated for 6 d. On day 4, cells were transfected with inducible Plk4-EGFP alone or with Asl-V5 and then induced to express the next day for 24 h. Proteins were detected by immunoblotting cell lysates with anti-GFP, V5, and SAS-6 antibodies. Cotransfected Nlp-EGFP was used as a loading control. Con, control. (C) Asl stabilization of Plk4 requires PB1-PB2. The relative protein stability of a Plk4-EGFP mutant lacking PB1-PB2 was analyzed by immunoblotting lysates of S2 cells that were or were not transiently coexpressing Asl-V5. Cotransfected Nlp-EGFP was used as a loading control. (D) PB1-PB2 is necessary for Plk4 to associate with Asl-C. S2 cells were control or Asl 5′–3′UTR RNAi treated for 7 d. On day 5, cells were cotransfected with inducible Asl-C–V5 and the indicated inducible Plk4-EGFP construct (or control EGFP) and induced to express the next day for 24 h, and then lysates were prepared for anti-GFP immunoprecipitation. Immunoblots were probed for V5, GFP, and endogenous Asl. IP, immunoprecipitation. (E) Asl-A and Asl-C interact specifically with PB1-PB2 in Plk4 by Y2H analysis. FL and fragments of Asl and Plk4 were screened by Y2H. In each image, colonies from replica plating are shown. (left image) Growth indicates the presence of both bait and prey. (right image) Growth on DDOXA, and color indicates an interaction. AA indicates that one or both protein fragments autoactivated the Y2H reporters on their own and could not be tested. Both Asl-A and Asl-C strongly interacted with PB1-PB2 but not PB3. KinDom, kinase domain.

Asl contains a second Plk4 PB1-PB2–binding domain in its C terminus that is sufficient for stabilizing Plk4. (A) Asl stabilizes Plk4 primarily through its C terminus. The relative stability of Plk4-EGFP protein was analyzed by immunoblotting lysates of S2 cells transiently coexpressing the indicated inducible Asl-V5 constructs. Cotransfected Nlp-EGFP was expressed under its endogenous promoter and served as a loading control. Blots were probed with anti-GFP, V5, and Asl. In lane 2, FL Asl-V5 and endogenous (Endo) Asl are not resolvable, so the band contains both species. (B) Asl stabilizes Plk4 in cells lacking centrioles. S2 cells were control or SAS-6 RNAi treated for 6 d. On day 4, cells were transfected with inducible Plk4-EGFP alone or with Asl-V5 and then induced to express the next day for 24 h. Proteins were detected by immunoblotting cell lysates with anti-GFP, V5, and SAS-6 antibodies. Cotransfected Nlp-EGFP was used as a loading control. Con, control. (C) Asl stabilization of Plk4 requires PB1-PB2. The relative protein stability of a Plk4-EGFP mutant lacking PB1-PB2 was analyzed by immunoblotting lysates of S2 cells that were or were not transiently coexpressing Asl-V5. Cotransfected Nlp-EGFP was used as a loading control. (D) PB1-PB2 is necessary for Plk4 to associate with Asl-C. S2 cells were control or Asl 5′–3′UTR RNAi treated for 7 d. On day 5, cells were cotransfected with inducible Asl-C–V5 and the indicated inducible Plk4-EGFP construct (or control EGFP) and induced to express the next day for 24 h, and then lysates were prepared for anti-GFP immunoprecipitation. Immunoblots were probed for V5, GFP, and endogenous Asl. IP, immunoprecipitation. (E) Asl-A and Asl-C interact specifically with PB1-PB2 in Plk4 by Y2H analysis. FL and fragments of Asl and Plk4 were screened by Y2H. In each image, colonies from replica plating are shown. (left image) Growth indicates the presence of both bait and prey. (right image) Growth on DDOXA, and color indicates an interaction. AA indicates that one or both protein fragments autoactivated the Y2H reporters on their own and could not be tested. Both Asl-A and Asl-C strongly interacted with PB1-PB2 but not PB3. KinDom, kinase domain.

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