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. Author manuscript; available in PMC: 2019 Oct 14.
Published in final edited form as: Cell Rep. 2018 Feb 20;22(8):2160–2175. doi: 10.1016/j.celrep.2018.01.080

Figure 7. Rap1 Interacts with Hpo to Suppress Its Kinase Activity.

Figure 7.

(A) A schematic diagram of full-length Hpo and its deletion constructs used in this study.

(B) SDS-PAGE gel stained with Coomassie blue showing the bacterially expressed MBP fusion proteins. Rap1 recombinant proteins are incubated in cell lysate containing FLAG-Hpo, and the precipitated complexes were subjected to western blotting (WB) with anti-FLAG antibody.

(C) WB with anti-MBP antibody revealing which of the Hpo domains pulls down MBP-Rap1V12.

(D) Hpo autophosphorylation is analyzed by immunoblot with anti-Hpo-pThr195 antibody, and the optical density ratio is normalized with FLAG-Hpo protein (α-FLAG). *p < 0.5, **p < 0.01. N.S. means not significant.

(E) Model of the Rap1-Hpo pathway in BC polarization. At the front of migratory clusters, GTP-bound Rap1 binds to Hpo to prevent its activation, relieving Ena/VASP suppression and promoting actin polymerization to advance BC migration. The error bars represent SD.