Fig. 7. Mutation of Tyr14 on caveolin inhibits the effects of P25 on fibronectin matrix assembly and β1-integrin activation. (A) MEF caveolin^+/+ (cav^+/+) and MEF cav^–/– cells infected with adenovirus containing wild-type (WT) or mutant (Y14F) FLAG-tagged caveolins were lysed and analyzed for caveolin expression by western blot. Blots were stripped and re-probed for FLAG to visualize ectopic expression of caveolins. (B) Monolayers of normal MEFs (cav^+/+), caveolin-null cells (cav^–/–) and caveolin-null cells infected with wild-type caveolin virus (WT) or caveolin Y14F mutant virus (Y14F) for 24 hours were incubated with 50 µM of either S25 or P25 in the presence of ¹²⁵I-fibronectin (¹²⁵I-FN) for 6 hours. Cell layers were rinsed in PBS and solubilized in 1 N NaOH. Radioactivity was measured by scintillation. (C) Cells were fixed and incubated with 9EG7, a monoclonal antibody against the activated β1 integrin, for an additional 1 hour. Bound antibody was detected by ELISA. The data are presented as fold increase over control, where the S25 value serves as the control for each experiment. Error bars indicate s.e.m. of triplicate samples. Asterisk (*) indicates statistical difference in effects of P25 on cells expressing wild-type caveolin versus cells expressing either no caveolin or mutant caveolin (t-test, P<0.05).