FIG. 4.

Enhancement of neutrophil elastase corepressor activity through nuclear localization. (A) Preferential localization of a neutrophil elastase N95A/N144A glycosylation variant in the nucleus. Shown is indirect immunofluorescence localization by confocal microscopy of neutrophil elastase (NE) (green) on the right, with DAPI (4′,6′-diamidino-2-phenylindole) (blue) nuclear and granular LAMP1 (red) counterstaining on the left column, for the wild type (top row) or a N95A/N144A glycosylation variant (bottom row) in transiently transfected RBL1 cells. (B) Transient-transfection reporter assay in NIH 3T3 cells showing better Gfi1 corepression with a nuclear-localizing glycosylation variant of neutrophil elastase. Cells were transfected with Gfi1-responsive CAT reporter or Gfi1 expression vector (45 ng), and the mass ratios of wild-type neutrophil elastase or neutrophil elastase N95A/N144A expression vector (both with the carboxyl-terminal propeptide intact) are indicated. CAT activity was normalized to cotransfected luciferase, and the total quantity of transfected DNA was held constant by the addition of a β-galactosidase expression vector. P values were determined by a two-tailed t test. **, P < 0.01; *, P < 0.05. The error bars indicate standard errors of the means.