Pleckstrin-2 colocalizes with F-actin at the immune synapse. (A) Conjugates were formed between SEE-pulsed Raji B cells stained with CMAC-7 (blue) and Jurkat T cells transfected with the various GFP-fused pleckstrin-2 constructs (green). The fixed conjugates were then stained with rhodamine-phalloidin (red) to label F-actin and analyzed by indirect immunofluorescence at 63× magnification. The merger of the red and green fluorescence to yellow demonstrates that pleckstrin-2 colocalizes with actin at the immune synapse. Simultaneous mutations in both PH domains prevented pleckstrin-2 from colocalizing with actin. Note that the cells are plated on poly-L-lysine and do not demonstrate membrane localization of pleckstrin-2. Data shown are from 1 representative experiment of 3 independent experiments. (B) Colocalization of pleckstrin-2 and actin was quantified by randomly selecting conjugates containing a green T cell contacting a blue B cell. Shown is the mean ± standard deviation of 3 independent experiments. The paired Student t test was performed of WT without SEE stimulation or the pleckstin-2 mutants compared with WT pleckstrin-2. *P < .05; **P < .005.