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. 2014 Jul 8;5(15):6142–6167. doi: 10.18632/oncotarget.2178

Fig.4. Inhibition of AKT promotes enhanced MDM2 activity via the increased association between NPM and p14ARF.

Fig.4

(A) Npm−/−, p53−/−double null MEF were infected with pBABE retrovirus empty vector and pBABE expressing FLAG-tagged-NPM-WT, NPM-S48A or S48E as indicated. Immunopurification of NPM was done by pulling down with the Flag tag (middle panel) followed by elution of complexes by the Flag peptide and subsequent immunopurification of endogenous MDM2 (lower panel). (B) Nuclear immunoprecipitates of MDM2 from T24 cells treated with MK-2206 (5 μM, 24 hrs). Immunoprecipitates and lysates were blotted with the indicated antibodies. (C) T24 cells were treated with MK-2206 (5 μM) as indicated. p14ARF was immunoprecipitated from whole cell lysates and nuclear extracts and the association with NPM and MDM2 determined by western blot. Immunoprecipitates and lysates were blotted with the indicated antibodies. (D) MDM2 and (E) p53 ubiquinitation assay in H1299 cells transfected with wild type p53, HA-tagged ubiquitin and treated for 16 hrs with DMSO, MK-2206 (5 μM) or Nutlin3A (5 μM) as indicated. Immunoprecipitates and whole cell lysates were probed with the indicated antibodies.