The nuclear protein import assay in vascular smooth muscle cells.

ABSTRACT

Nuclear protein import plays a critical role in both proliferation and apoptosis. Both actions are of great importance in vascular development and pathology. Consequently, a technique that would allow us to characterize import and identify novel cofactors important in modulating the process in smooth muscle cells is of great applicability. In this article, we describe a simple and reliable nuclear protein import assay that we have modified for use on aortic smooth muscle cells in culture. Briefly, the procedure permeabilizes the cells and analyzes the nuclear retention of a fluorescent import marker. Using this method, we are able to analyze the effect of agents on nuclear protein import. Most importantly, we are able to treat the cytosol, nucleus or the whole cell independently. This technique will allow for the identification and development of drugs to inhibit or stimulate the process and, potentially, to identify mechanistic insight into disease processes.