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Figure 3: Analysis of NCSTN domain functions. (a) Structures of wild-type (WT) and mutant NCSTN. Deletion mutants of NCSTN were constructed as described in the Materials and methods. Deleted regions in NCSTN $\mathrm{\Delta }$195–305, NCSTN $\mathrm{\Delta }$306–360, NCSTN $\mathrm{\Delta }$361–418, NCSTN $\mathrm{\Delta }$ 419–458 and NCSTN $\mathrm{\Delta }$625–662 are shown. SP and TM represent the signal peptide and the transmembrane domain, respectively. Striped regions between amino acid positions 306 and 360, 419 and 458, and 625 and 662 are highly conserved sequences amongst NCSTN orthologs. (b) Comparable expression levels of NCSTN in mutant-NCSTN transfectants. ras-NIH3T3 cells were transfected with cDNA of wild- type or mutant NCSTN. Cells were harvested 48 hours after transfection, and the NCSTN protein expression levels were examined by Western blotting. mNCSTN and hNCSTN represent endogenous mouse NCSTN and ectopically expressed human NCSTN, respectively. (c) Effects of NCSTN mutants on the p53-responsive promoter. ras-NIH3T3 cells were cotransfected with p53-responsive reporter plasmid pG13-Luc and the expression plasmid for wild-type or mutant NCSTN. Cells were harvested 48 hours after transfection and the luciferase activities in the cell extracts were measured. The error bars represent SD ($n=3\text{,}$ t-test; *$P<.01$).