Figure 4.

Role of p130^CAS and Crk in integrin-mediated activation of JNK. 293 cells were transiently transfected with 3 μg of vector encoding FLAG-tagged JNK alone or in combination with 3.75, 7.5, and 15 μg of plasmid encoding a mutant form of p130^CAS carrying a deletion of the entire substrate region (SD-CAS), or with 3, 9, and 27 μg of plasmids encoding SH2⁻ v-Crk (SH2 mutant), SH3⁻ v-Crk (SH3 mutant), and GST-DnMKK4 (kinase inactive). The cells were serum starved for 24 h, detached, and solubilized immediately (S) or plated on fibronectin for 20 min (Fn). FLAG-tagged JNK was immunoprecipitated from extracts containing equal amounts of total proteins and subjected to an in vitro kinase assay with GST-Jun as a substrate (A). Total lysates containing the same amount of proteins were subjected to immunoblotting with antibodies to p130^CAS, gag, and GST to verify that the expression of the various dominant-negative proteins was proportional to the amount of DNA transfected (B). Immunoblotting with anti-FLAG antibodies was used to verify equal expression of FLAG-JNK in all samples (C).