Figure 5.

The E-box sequence is essential for TFE3-mediated and TGF-β-dependent activation of the PE2 fragment of the PAI-1 promoter. (A) The sequence of the PE2 promoter. (B) Hep G2 cells were transfected with 1 μg of PE2–Luc or PmE2–Luc DNA (CACGTG → acCGac), 1 μg of pSVβ, and also 1.0 μg of plasmid encoding TFE3 as indicated. Transfected cells were treated with (█) or without (□) TGF-β, processed, and assayed as described in Fig. 3B. (C) TFE3 was synthesized in vitro from pET–TFE3 by the TNT T7 Coupled Reticulocyte Lysate System (Promega). Gel-shift reactions contained 3 μl of the in vitro TFE3 translation reaction and 1 μl of (4 × 10³ cpm) of ³²P-labeled PE2 DNA probe. Reactions in lanes 3 and 4 contained a 50-fold excess of wild type or mutant PE2 oligonucleotides, respectively.