Fig. 4. Coupled ERK immunoprecipitation/MBP kinase assay for assessment of TGF-ß1-induced ERK activation. ERK1/2 were immunoprecipitated from lysates of quiescent (Q), FBS- and TGF-ß1-stimulated T2 cells. Exposure of quiescent cells to FBS (20%) was for 15 minutes and stimulation with TGF-ß1 (1 ng/ml) was for 30, 60 and 90 minutes prior to cell disruption and ERK1/2 immunoprecipitation. MBP phosphorylation reaction products (MBP-P) were separated by gel electrophoresis; equivalent loading of MBP and ERK per lane was confirmed by Ponceau S staining (not shown) and ERK2 western blotting, respectively (A). In contrast to the relatively rapid rate of ERK activation by serum (15 minutes), TGF-ß1-induced changes in ERK activity were not evident until 60 minutes after growth factor addition (A), remained elevated for approximately 2 hours and then rapidly declined (B). Coupled ERK immunoprecipitation/MBP phosphorylation (MBP-P) assays (C) confirmed that ERK activation in TGF-ß1-stimulated T2 cells is sensitive to the same pharmacologic inhibitors that attenuate growth factor-induced PAI-1 expression. The more pathway restrictive inhibitor herbimycin A (250 nM) (Fukazawa et al., 1994) attenuated MBP phosphorylation but not to the same extent as genistein or PD98059.
