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. 2015 Jun 29;112(28):8632–8637. doi: 10.1073/pnas.1510929112

Fig. S6.

Fig. S6.

Additional in vivo ubiquitination experiments related to Fig. 4. (A) In vivo ubiquitination assay. Extracts from MCF7 cells treated in the presence or absence of KU-55933 and expressing Flag-MDM2, myc-FBXO31, and HA-ubiquitin (HA-Ub) were immunoprecipitated with a Flag antibody and probed for HA-ubiquitin. GFP expressed from a cotransfected plasmid was used as transfection/loading control. (B and C) In vivo ubiquitination assays. H1299 cells were transfected with plasmids expressing His-ubiquitin, Flag-MDM2, either myc-FBXO31 or myc-FBXO31∆F, and either GFP alone (B) or GFP-p53 (C). Proteins bound to His-ubiquitin were purified by Ni-NTA pull-down, washed, and eluted in imidazole. Ubiquitinated Flag-MDM2 and GFP-p53 were detected using an anti-Flag or anti-GFP antibody, respectively.