Fig. 8. CD-mediated PAI-1 expression and ERK1/2 activation is MEK dependent. Quiescent R22 cells were pretreated with PD98059 (at the indicated concentrations) prior to addition of CD. Northern blots of total cellular RNA were probed with ³²P-labeled cDNA probes to PAI-1 and A50; PAI-1 mRNA abundance was normalized to A50 signal (A). CD-induced ERK1/2 activity was assessed by a coupled immunoprecipitation—in-vitro-kinase assay using myelin basic protein (MBP) as a phosphorylation substrate with or without PD98059 pretreatment (B). Western blotting and Ponceau S staining (B) served to confirm ERK1/2 and MBP levels, respectively, for normalization of MBP phosphorylation activity. The histograms (A,B) illustrate results of three separate experiments (mean±s.d.). Untreated groups in both panels refer to DMSO vehicle only.