Transfection and Immunofluorescence Assays.
Plasmid DNA for expression constructs containing PSTPIP1, PTP-PEST, WASp, WASpY291F, and WASpY102F cDNAs were purified using CLONTECH Laboratories, Inc. Maxi-Prep kit. 5 × 104 Cos-7 cells maintained in DMEM supplemented with 10% fetal bovine serum, l-glutamine, and penicillin/streptomycin, were seeded onto glass coverslips and transfected with selected plasmid DNA using Lipofectamine 2000 (Invitrogen). At 24 h after transfection, cells were washed and fixed with 3% ice-cold paraformaldehyde in PBS. Alternatively, unstimulated or stimulated OT-II T cells were transfected by electroporation (1 pulse, 360 mV) using a BTX electroporator and subjected to fixation at 2.5 h after transfection. After fixation, cells were blocked with 2% BSA/PBS for 10 min immediately or for intracellular staining, cells were first permeabilized with 0.1% Triton X-100/PBS. Cells were then incubated with primary and the appropriate fluorescently conjugated secondary antibodies and the stained samples were mounted in anti-fade mounting media (DakoCytomation). Images were analyzed using the Olympus 1X-70 inverted microscope equipped with fluorescence optics and Deltavision Deconvolution Software (Applied Precision).