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. 2014 Oct 31;5(20):9756–9769. doi: 10.18632/oncotarget.2386

Figure 5. AM inhibits the interaction between MDM2 and p53 in the nucleus.

Figure 5

(A) A ubiquitination assay was performed to compare the effects of AM and PBS on cells. IEC-6 cells were transfected with HA-tagged ubiquitin and then treated with MG132 (20 μg/mL) for 4 hours to inhibit proteasome function. AM or PBS was then administered for 60 minutes. Western blots of p53 were performed to detect polyubiquitination of p53. (B) Immunoprecipitation (IP) for MDM2 and subsequent immunoblotting (IB) for p53 was performed using total cell lysates. IEC-6 cells treated with AM or PBS for 60 minutes were used for IP and IB. (C) IP for p53, followed by IB for MDM2 or NQO1, was performed using total cell lysates. (D) In situ PLA for p53 and MDM2 20 and 60 minutes after administration of AM or PBS.