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. Author manuscript; available in PMC: 2017 Mar 4.
Published in final edited form as: Mol Cell. 2015 Feb 26;57(6):1022–1033. doi: 10.1016/j.molcel.2015.01.015

Figure 2. Skp2-SCF ubiquitinates LKB1 via K63-linkage.

Figure 2

(A, B) IPs by anti-LKB1 (A) or anti-Skp2 (B) antibody from HEK293 cells with control, Skp2 or LKB1 knockdown were subjected to IB. The asterisks indicate heavy chains of the antibodies and the arrowheads indicate the bands corresponding to Skp2 (A) or LKB1 (B).

(C, D, F) In vivo ubiquitination assays in HEK293T cells (C and F) or HEK293 cells with control or Skp2 knockdown (D) transfected with the indicated plasmids were followed by IB. Ni-NTA, nickel bead precipitates.

(E) In vitro ubiquitination assay, in which recombinant GST-LKB1 was incubated with purified Flag-Skp2/SCF along with recombinant ubiquitin (Ub), E1 and E2 enzymes for reaction, was followed by IP and IB.

(G) IPs by anti-IgG control or anti-K63-Ub antibody from HEK293T cells transfected with the indicated plasmids were subjected to IB.

See also Figure S2.