Figure 3.

Effect of TGF-β on the PAI-1 mRNABp–PAI-1 mRNA binding interaction. (A) MeT5A cells were treated with PBS or TNF-α (10 ng/ml) or TGF-β (2 ng/ml) for 24 hours at 37°C in serum-free RPMI media. The cytosolic extracts were prepared and separately incubated with ³²P-labeled PAI-1 mRNA CDR or 3′UTR in the presence of tRNA. The reaction mixtures were later digested with RNase T1 and heparin as described in Materials and Methods to avoid nonspecific interaction. After heparin digestion, the reaction mixtures were separated on native polyacrylamide gel (4%), dried, and autoradiographed. Fp = free probe. (B) Specificity of the PAI-1 mRNABp–PAI-1 mRNA 3′UTR interaction. Cytosolic extracts of MeT5A cells were incubated with varying amounts of unlabeled transcript before exposure to ³²P-labeled transcript. The reaction mixtures were later digested with RNase T1 and heparin and UV irradiated at 4°C. The immobilized RNA–protein complexes were separated on a SDS-PAGE, dried, and autoradiographed. (C) Cytosolic extracts from MeT5A, M33K, M9K, and MS-1 cells were subjected to PAI-1 mRNA 3′UTR binding analyses as described in (A) in the presence of tRNA. After RNase T1 and heparin digestion, the reaction mixtures were subjected to UV cross-linking and autoradiography. The panels are representative of two independent repetitions.