Effect of insulin on PAI-1 expression by cultured 3T3-L1 adipocytes
3T3-L1 adipocytes were grown and differentiated in 100-mm tissue culture plates as described (36). Total RNA was isolated from untreated cells (○), and cells treated with 100 nM insulin (●) for the indicated times, and the steady-state levels of PAI-1 mRNA were determined using quantitative PCR (n = 6 ± SD). Inset: Conditioned medium (20 µl) from untreated cells and cells treated with insulin for various times were electrophoresed under non-reducing conditions on a 9% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were analyzed for PAI-1 by Western blotting using a polyclonal rabbit anti-mouse PAI-1 antiserum and the enhanced chemiluminescence detection system. mPAI-1 refers to the recombinant murine PAI-1 antigen used as a standard.