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. Author manuscript; available in PMC: 2013 Aug 7.
Published in final edited form as: Oncogene. 2012 Mar 5;32(3):388–396. doi: 10.1038/onc.2012.63

Figure 3. RPS14 stabilizes p53 by inhibiting MDM2-mediated p53 ubiquitination.

Figure 3

(A) Ectopic expression of RPS14 inhibits MDM2-mediated p53 ubiquitination in cells. H1299 cells were transfected with combinations of plasmids encoding p53, HA-MDM2 or Flag-RPS14 in the presence of the His-Ub plasmid. Cells were treated with MG132 for 8 h before harvesting. Cell lysates were subjected to an ubiquitination assay followed by IB with anti-p53. The expression levels of p53, HA-MDM2 and Flag-RPS14 are shown in the lower panels. (B) Ectopic expression of RPS14 stabilizes exogenous p53 in H1299 cells. Cells were transfected with combinations of p53, HA-MDM2 or Flag-RPS14 in the presence of pEGFP plasmids as a control and harvested 36 h post-transfection for IB with antibodies as indicated. (C) RPS14 stabilizes endogenous p53 in A549 cell. Cells were transfected with an increasing amount of the Flag-RPS14 plasmid and harvested 36 h after transfection for IB with antibodies as indicated. (D) RPS19 does not stabilize endogenous p53 in A549 cell. The assay as that in panel C was conducted except the RPS19 plasmid was used here. (E) RPS14 extends the half-life of endogenous p53. A549 cells were transfected with pcDNA3 or Flag-RPS14 and treated with cycloheximide (CHX) at 36 h post-transfection, and harvested at different time points for IB with antibodies as indicated. (F) A graphic presentation of the result from panel E. (G) wild-type RPS14 and its C-terminus (amino acids 51–151), but not its N-terminus (aa 1–51), drastically induced p53 in A549 cells. Cells were transfected with pcDNA3, Flag-RPS14/1–50, Flag-RPS14/51–151, or Flag-RPS14 plasmids and harvested 36 h after transfection for IB with antibodies as indicated. (H) RPS14 slightly induces a p53 apoptotic target, Bax, but not the cleaved-Parp. A549 cells were transfected with pcDNA3 or Flag-S14 plasmids and harvested for IB with antibodies as indicated.