Figure 3.

Functional and Western immunoblot analyses of mutant IPF-1 proteins. (a) Transactivation assays were done in HIT-T15 insulinoma cells that express endogenous IPF-1. Cells were transiently transfected with a rat insulin-promoter-CAT reporter plasmid and 0.3–33 ng of expression plasmids for wild-type IPF-1 (WT) or the mutant IPF-1 isoforms. The number of independent experiments is 13–14, done on 2–3 different occasions. Basal CAT activities ranged from 6% to 15% conversion to acetylated forms of chloramphenicol. Mean values ± SEM are given compared to basal values (transfection with empty vector) normalized for the independent experiments. P values, shown above the bars (± SEM), were determined by the Wilcoxon rank sum test comparing the effect of each mutation to wild-type IPF-1 (WT). For the same comparisons, P values using the unpaired 2-sided Student’s t test were 0.052, 0.28, and < 0.001 for D76N, Q59L, and InsCCG243, respectively. (b) Determination of IPF-1 expression levels by Western immunoblot using antiserum α253 (14), specific for the detection of the COOH-terminal region of IPF-1: 500 ng of IPF-1 expression plasmids was transfected to the HIT-T15 cells. The relative expression levels of the wild-type (lane 2), Q59L (lane 4), and InsCCG243 (lane 5) IPF-1 forms were the same. The D76N form (lane 3) was expressed at a lower level than that of the others. After correction for expression efficiency by Western immunoblot analysis, the D76N mutant function was approximately 60% that of the wild-type IPF-1. Lane 1 corresponds to extracts from cells transfected with the empty vector.