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. 2022 Jul 19;10:922675. doi: 10.3389/fcell.2022.922675

FIGURE 2.

FIGURE 2

MAGE-C2 directly interacts with MDM2 and inhibits its ligase activity through the MHD domain. (A) Interactions between MAGE-C2 and MDM2 were validated by using GST pulldown assay. The indicated GST- and SUMO-tagged proteins were expressed in bacterium, and SUMO–MDM2 proteins were detected by using anti-MDM2 Western blot. GST was included as an irrelevant negative control protein. (B) Interactions between MAGE-C2 mutants and MDM2 were validated by using GST pulldown assay. The indicated GST-tagged MAGE-C2 proteins were incubated with in vitro translated SUMO–MDM2. Proteins were detected by using anti-MDM2 immunoblotting. (C) In vitro ubiquitination assay. His-ubiquitin, ubiquitin E1, UbcH5B E2, Mg-ATP, GST-p53, and the indicated MAGE-C2 and MDM2 proteins were incubated for specific time at 37°C and detected by using anti-p53 immunoblotting. (D) Ubiquitination assay in cells. HEK293T cells were transfected with HA-ubiquitin and indicated plasmids. The level of p53 ubiquitination was determined by using anti-Flag M2-agarose redbeads purification and anti-HA Western blot.