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. Author manuscript; available in PMC: 2020 Dec 17.
Published in final edited form as: Oncogene. 2020 Jun 17;39(29):5228–5239. doi: 10.1038/s41388-020-1359-4

Figure 3. Neddylation of VHL by Mdm2 interferes with VHL-p53 complex formation.

Figure 3.

(A) Transient transfection in H1299 cells under normoxia or hypoxia. Cell lysates were immunoprecipitated for HA and subjected to analysis by Western blot.

(B) Immunoprecipitation of VHL from 786-O cells treated with the neddylation inhibitor MLN4924 and Western blotted for p53 and VHL.

(C) Immunoprecipitation of p53 or IgG control from MCF7 cells treated with MLN4924 for various indicated times and Western blotted for VHL and p53.

(D) GST pulldown of GST, GST-alpha VHL, GST-beta VHL, or GST-VHL with His-Mdm2 and Western blotted for GST and Mdm2.

(E) Cell free neddylation assay using recombinant proteins. Reactions were separated by SDS-PAGE and analyzed by Western blot.

(F) Immunoprecipitation of endogenous nedd8 from 786-O cells transfected with Mdm2 and subjected to hypoxia and Western blotted for VHL and Mdm2.

(G) Ni-NTA pulldown for His-nedd8 in H1299 cells in a transient transfection and subjected to treatment with MLN4924. Samples were separated with SDS-PAGE and analyzed by Western blot for VHL and Mdm2.

(H) Schematic of the p53-pVHL-Thrombospondin pathway disrupted by c-Src-Mdm2.