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. 2010 Nov 20;39(6):2234–2248. doi: 10.1093/nar/gkq1117

Figure 5.

Figure 5.

PAK1IP1 associates with MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. (A) Ectopically expressed PAK1IP1 interacts with ectopically expressed MDM2 in 293T cells. Cells were transfected with Flag-PAK1IP1 and pcDNA3-MDM2 individually or together. Cell lysates were immunoprecipitated with anti-Flag or anti-MDM2 antibodies, followed by IB with anti-Flag or anti-MDM2 antibodies. (B) Endogenous PAK1IP1 interacts with endogenous MDM2 in U2OS cells. Cell lysates were immunoprecipitated with anti-PAK1IP1 polyclonal antibodies or rabbit immunoglobulin G (IgG), anti-MDM2 antibodies, or IgG, followed by anti-PAK1IP1 and anti-MDM2 antibodies, respectively. (C) PAK1IP1 colocalizes with MDM2 in the nucleus. U2OS cells were co-transfected with GFP-PAK1IP1 and Flag-MDM2. The cells were observed with a confocal laser scanning microscopy for GFP (green) or were immunostained with anti-Flag antibody, followed by staining with goat anti-rabbit secondary antibody (red) and DAPI. (D) PAK1IP1 inhibits MDM2-mediated p53 degradation. H1299 cells were transfected with the combinations of plasmids as indicated. Cell lysates were subjected to immunoblot using antibodies as indicated. (E) PAK1IP1 inhibits MDM2-mediated p53 ubiquitylation. H1299 cells were transfected with the combinations of indicated plasmids. The transfected cells were treated with MG132 (20 μM) for 6 h before harvest. Total p53 and ubiquitylated proteins p53 [p53-(ub)n] were detected by immunobloting with specific anti-p53 antibody (upper panel). Ubiquitylated was indicated. The expression of total p53, MDM2, and PAK1IP1 proteins were shown in the lower panels. Bars, 10 μm.