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. Author manuscript; available in PMC: 2017 Sep 27.
Published in final edited form as: Oncogene. 2017 Mar 27;36(30):4299–4310. doi: 10.1038/onc.2017.64

Figure 4. Skp2 interacts with and promotes non-degradative ubiquitination of Twist.

Figure 4

(a) PC3 cells were used for immunoprecipitation (IP) with Twist antibody followed by western blot analysis. (b) In vivo ubiquitination assay in 293T cells transfected with Xpress (Xp) tagged Skp2, Histidine-tagged ubiquitin (His-Ub) and Flag-tagged Twist. Ni-NTA indicates nickel nitrilotriacetic acid bead precipitate; WCE indicates whole cell extract. (c) In vivo ubiquitination assay in 293T cells transfected with various plasmids. (d) In vivo ubiquitination assay in 293T cells transfected with Xp-Skp2 and Flag-Twist along with His-Ub WT, His-Ub K63R or His-Ub K48R constructs. (e) 293 cells with GFP or Skp2 knockdown transfected with Flag-tagged β-TrCP and Xp-tagged Twist were subjected to IP after 48 hours with Flag antibody followed by western blot analysis. The relative intensities of immunoprecipitated Twist were quantified with Image J software and normalized with Twist in the input.