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. 2016 Feb 18;7(13):16217–16226. doi: 10.18632/oncotarget.7479

Figure 2. Overexpression of RPL4 inhibits MDM2-mediated p53 ubiquitination and degradation.

Figure 2

A. Overexpression of RPL4 inhibits MDM2-mediated p53 degradation. H1299 cells were transfected with the indicated plasmids and assayed for the expression of p53 and MDM2 by IB. B. RPL4 inhibits MDM2-mediated p53 ubiquitination in cells. H1299 cells transfected with combinations of the indicated plasmids were treated with MG132 (40 μM) for 6 hours before harvesting, followed by the in vivo ubiquitination assay. The ubiquitinated species of p53 detected by IB using the anti-p53 antibodies are indicated on right of upper panel. The expression of indicated proteins is shown in lower panels. C. Overexpression of RPL4 induces the levels of endogenous p53 in cells. U2OS cells transfected with the increasing amount of Flag-RPL4 plasmid were assayed by IB. D, E. Induced expression of RPL4 also stabilizes p53. U2OS-TO-Flag-RPL4 cells expressing tetracycline (tet)-inducible Flag-RPL4 (clone 2) were cultured in the absence or presence of doxycycline (2 μg/ml) for different time points, followed by IB detection of the indicated proteins (D). IB was also performed in three individual U2OS-TO-Flag-RPL4 clones cultured in the absence or presence of doxycycline (E). F, G. Overexpression of RPL4 stabilizes p53. U2OS cells transfected with control or Flag-RPL4 were treated with 50 μg/ml of CHX and harvested at different time points. The cell lysates were assayed by IB to detect the levels of the indicated proteins (F). The relative levels of p53 were normalized against the expression of actin and plotted in G. H. Overexpression of RPL4 induces the mRNA levels of p53 target genes. U2OS-TO-Flag-RPL4 cells were cultured in the absence or presence of doxycycline (2 μg/ml) for 24 hours. The cells were assayed for mRNA expression of the indicated genes by RT-qPCR, normalized to the expression of GAPDH.