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. 2012 Mar 12;287(18):14621–14630. doi: 10.1074/jbc.M111.301523

FIGURE 4.

FIGURE 4.

SKI regulates MDM2 through sumoylation to result in a decrease of endogenous p53. A, SKI can enhance MDM2 sumoylation through PIAS1. 24 h after expression, plasmids were introduced into 293T cells, and cells were lysed in RIPA buffer, and the supernatant was diluted into denaturing buffer. The MDM2 was isolated through nickel-nitrilotriacetic acid (NTA) beads followed by immunoblot (IB) analysis with anti-FLAG antibody to reveal MDM2 sumoylation. The blot was stripped and probed with anti-MDM2 antibody to reveal the MDM2 level. Separate lysate was used to examine PIAS1 and SKI expression levels using anti-PIAS1 and anti-SKI antibody, respectively. B, SKI can enhance MDM2 sumoylation through PIAS3. 40 h after transfection, cells were processed as in A. A portion of the lysate was analyzed to gauge the protein expression through immunoblot with anti-PIAS3 and anti-SKI antibody. C, SKI mutant ARPG fails to increase MDM2 sumoylation. 40 h after transfection, cells were processed as in A. A portion of the lysate was analyzed to gauge the protein expression through immunoblot with anti-PIAS3 and anti-SKI antibody. D, SKI enhances p53 ubiquitination through PIAS1 and -3. H1299 cells were transfected with various combinations of expression plasmids as well as siRNAs. The cells were processed as in Fig. 2. The effectiveness of siRNA for PIAS1 or -3 is shown in E. E, SKI and PIAS1 or -3 are required for maintaining the p53 level in LNCaP cells. Various siRNAs were introduced into LNCaP cells, and 40 h later, cells were lysed, and endogenous p53 as well as other proteins were examined by immunoblot analysis. The effectiveness of siRNA for PIAS1 or -3 was shown by the reduction of PIAS1 or -3 in 293T cells transfected with the expression plasmid alone or with the respective siRNA shown in the lower panel. F, down-regulation of p53 by overexpression of SKI can be reversed by a reduction of PIAS3. Expression plasmids for SKI and siRNAs were introduced into HEK293 cells, and 40 h later, cells were lysed, and endogenous p53 and MDM2 were examined by immunoblot analysis.