Expression of pleckstrin-2 in Jurkat cells stimulates clustering of α4β1 and enhances adhesion to fibronectin. (A) Jurkat cells expressing GFP alone, WT pleckstrin-2, the PH domain mutant, or the DEP domain mutant were plated on fibronectin. Jurkat-cell adhesion was quantitated by measuring the activity of acid phosphatase 32. Shown is the mean ± SEM of 6 independent experiments. The paired Student t test was performed of the transfectants compared with WT pleckstrin-2. *P < .005 (B) GFP-fused pleckstrin-2 constructs (green) were plated on fibronectin, fixed, stained with a monoclonal antibody to the integrin β1 subunit (red), and analyzed by indirect immunofluorescence. The merger of the red and green fluorescence to yellow demonstrates that pleckstrin-2 colocalizes with β1. Simultaneous mutations in both PH domains prevented pleckstrin-2 from colocalizing with β1. Images were captured using an UtraVIEW-LCI confocal scanner at ×100 magnification. Data shown are representative of 3 independent experiments. (C) The same GFP-fused pleckstrin-2 constructs, stained with a monoclonal antibody to the integrin β1 subunit, were plated on fibronectin and analyzed using total internal reflection fluorescence (TIRF). Cells transfected with WT pleckstrin-2 demonstrate enhanced β1 clustering at the point of adhesion to fibronectin. Images were captured using a Hamamatsu digital camera and Metamorph software at × 60 magnification. Data shown are representative of 3 independent experiments.