A mutant SWAP-70, SWAP-70-374, locates at the plasma membrane.

A, Binding activities of the mutant SWAP-70s to PtdIns(3,4,5)P₃ were analyzed using the bead binding assay. The upper panel shows SWAP-70s bound to the beads and the lower panel shows the protein used for the assay. B, Expression vectors for the wild-type while the mutant SWAP-70 were introduced into Cos7 cells and the localization of expressed proteins was analyzed by confocal microscopy. The stacked images are shown. The arrows indicate the cells whose SWAP-70 were found at the plasma membrane. C, The percentages of the cells whose GFP-SWAP-70s were found at the plasma membrane are shown. Experiments were repeated three times and the means are shown in the graph. The error bars show standard deviation. D, Expression vectors for GFP-SWAP-70 or GFP-SWAP-70-374 were introduced into Cos7 cells by eletroporation. The cells expressing GFP-SWAP-70 were stimulated with EGF (100 ng/ml) for 5 min. After fixation with formaldehyde, cells were stained with TRITC-phalloidin and observed using confocal microscopy. The upper panels show XY images of the cells and the lower XZ images. The lines in the top panels show the place where XZ images were made.