Figure 8.

Effect of alanine substitution in promoting nuclear localization of basonuclin. Human keratinocytes grown at low density in keratinocyte growth medium without 3T3 support were transfected with plasmid (pHUB19) encoding GFP basonuclin containing the mutations Ser-537-Ala, Ser-541-Ala. It was found that as the basonuclin overproduced from the plasmid was more brightly stained than the endogenous basonuclin, its localization could be more easily demonstrated by the antiserum to basonuclin than by the GFP tag. After an 18-hr incubation without 3T3 support in FAD medium (suboptimal conditions), the culture was fixed and stained for basonuclin and DNA. Under these conditions, endogenous basonuclin remains cytoplasmic. Wild-type basonuclin expressed from the plasmid is also cytoplasmic, whereas the mutant is strongly concentrated in the nucleus.