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. 2018 Jan 17;115(5):998–1003. doi: 10.1073/pnas.1705954115

Fig. 3.

Fig. 3.

Polyubiquitination of FBXO31 by CDH1 or CDC20 is dependent on phosphorylation by AKT kinase. (A and B) In vivo ubiquitination assay. HEK293T cells were cotransfected with plasmids expressing His-ubiquitin, Flag-FBXO31(WT), Flag-FBXO31(ΔD1), or Flag-FBXO31(R64A/L67A) and HA-CDH1 (A) or HA-CDC20 (B). Proteins bound to His-ubiquitin were purified by Ni-NTA pull-down, washed, and eluted in imidazole. Ubiquitinated Flag-FBXO31 was detected using an anti-Flag antibody. (C) Immunoblot monitoring the levels of FBXO31 and total AKT (t-AKT) in HEK293T cells expressing a NS shRNA or one of two unrelated AKT shRNAs. (D) Immunoblot monitoring FBXO31, phosphorylated AKT (p-AKT), and t-AKT in HEK293T cells treated with various concentrations of AKT inhibitor (AKT-I). (E and F) Immunoblot monitoring endogenous FBXO31 in HEK293T cells expressing HA-CDH1 (E) or HA-CDC20 (F) in the presence or the absence of AKT-I. (G) Coimmunoprecipitation monitoring the presence of phosphorylated Ser (p-Ser) or phosphorylated Thr (p-Thr) in the FBXO31 immunoprecipitate from HEK293T cells treated in the presence or the absence of AKT-I. (H) In vitro kinase assay monitoring the ability of purified AKT to phosphorylate GST-FBXO31(WT) or GST-FBXO31(S33A). (I and J) Immunoblot monitoring levels of Flag-FBXO31(WT) or Flag-FBXO31(S33A) in HEK293T cells expressing vector, HA-CDH1 (I), or HA-CDC20 (J).