Figure 2. SCF^FBXW7α ubiquitinates PLK1.

(A) In vitro ubiquitin ligation assay of ³⁵S labeled in vitro-transcribed/translated PLK1 was conducted in the presence or absence of the following products: cold in vitro-transcribed/translated SKP2, βTrCP, FBXW7αΔF or FBXW7α, E1 (His₆-ubiquitin activating enzyme), E2 (His₆-UbcH3 and UbcH5a) and Ub (ubiquitin). Samples were incubated at 30°C for 1h. The bracket on the left side marks a ladder of bands corresponding to poly-ubiquitinated PLK1. (B) The experiment was performed as in (A) except that the unlabeled F-box protein was substituted by a recombinant SCF^FBXW7α complex expressed in Sf21 insect cells. (C) HCT116 cells were transfected with plasmids encoding the indicated proteins, and treated with LLnL for 4h before harvesting. Extracts were prepared as indicated in the Materials and Methods and poly-ubiquitinated PLK1 visualized after Western blots of the PLK1 immunoprecipitations.